Medication adherence monitoring device

ABSTRACT

A Self Monitoring And Reporting Therapeutics, SMART® composition, method, apparatus and system are provided which flexibly provide options, by combining different embodiments of the device with different embodiments of the composition, the ability to conduct definitive medication adherence monitoring over the short term (Acute Medication Adherence Monitoring, immediately up to an hour or so after taking a medication), intermediate term (Intermediate Medication Adherence Monitoring, IMAM, an hour or so to a day or so after taking a medication), and longer term (Chronic Medication Adherence Monitoring, CMAM, a day to several days after taking a medication).

1.0 FIELD OF THE INVENTION

An improved Medication Adherence Monitoring System (MAMS) referred to asSMART®, an acronym for Self Monitoring and Reporting Therapeutics, isprovided comprising an optimized device, medication composition, andmethod of making and using the system and its components.

2.0 BACKGROUND OF THE INVENTION

As recently as 2012, it has been acknowledged in the literature (see,for example, Oberguggenberger et al., BMC Cancer, 2012, 12:474,“Adherence evaluation of endocrine treatment in breast cancer:methodological aspects”), that the assessment of long-term adherentbehavior with respect to medication regimens “is methodologicallychallenging. Studies have yielded inconclusive results indicatingadherence rates between 20% and 100% across different phases ofantineoplastic treatment. This variability of non-adherence rates foundin the literature has been suggested to be attributed to heterogeneousstudy designs as well as inconsistencies in methodological approaches.Among the latter the indirect methods of self-report, prescriptionrefill and pharmacy records have been predominately used in studies onadherence to endocrine agents. Direct methods which are supposed toreveal more objective results due to the assessment of medicationconsumption in an unmediated way have not been employed in respectivestudies. There is currently no Gold Standard of adherence measurementavailable [8].” Reference [8] to which Oberguggenberger et al., point insupport of these assertions is a 2003 WHO report entitled “ADHERENCE TOLONG-TERM THERAPIES—Evidence for action”. At page XIII of the report,the WHO articulated “Take Home Messages” which, in sum, stand for theproposition that there remains a long-felt need in the field ofmedication adherence monitoring that is currently not being adequatelymet by any available system.

Xhale, Inc., is a medical device development company which, for the lastseveral years, has been developing, improving and perfecting a state ofthe art Medication Adherence Monitoring System (MAMS). The improved(MAMS) according to this invention, referred to as SMART®, an acronymfor Self Monitoring and Reporting Therapeutics, is provided comprisingan optimized device, medication composition, and method of making andusing the system. The improved SMART® system according to this inventionprovides an integrated series of solutions to meet the long-felt needfor a reliable, gold-standard system to enable automated confirmation ofsubject adherence to a wide range of medication dosage regimens andcontexts.

Whereas various specific and general solutions have been reported in theart aimed at meeting this need, some of which are discussed below, thepresent patent disclosure for the provides an integrated system capableof providing definitive medication adherence assessments and monitoring,both on an acute (dose-to-dose) basis, and on the basis of longer timeframes, in the case of certain specific embodiments disclosed herein, upto and including over several doses of a given medication, over severaldays, or both. This is enabled by providing: a highly sophisticateddevice which includes heretofore unknown features and combinations offeatures for integrated use in combination with novel medicationcompositions, thus defining novel methods of utilizing the system toachieve medication adherence monitoring. Each of these elements of theintegrated system is taken up in turn in this patent disclosure, withextensive but non-limiting exemplary support, to enable and fullydescribe the various embodiments and equivalents thereof encompassed bythe present system.

Those skilled in the art will appreciate that the field of MAMS,including that of the SMART® system mentioned in art discussed hereinbelow, is typically an incremental process, as reflected in differentpublications and patent filings over an extended period of time. At acertain point of development, enough incremental advances on severalfronts coalesce, with a plurality of inter-related improvements havingbeen discovered in technical competence, enhancements in the apparatusesutilized to ask diverse questions which enable new methods to be appliedand tested. The present patent disclosure aspires to provide a detailedwritten description of what the current state of this technology nowenables.

To provide an adequate context for the plethora of advances found in thedetailed disclosure of the invention herein below, there is now provideda brief review of some key developments previously reported in thisfield, in related or competing fields, and, in some instances, inunrelated fields.

In 1999, a patent filing was conducted which ultimately led to issuanceof U.S. Pat. No. 7,820,108, for a “Marker detection method and apparatusto monitor drug compliance”, which generically disclosed and claimed amethod to determine whether a patient has taken a medication by:providing to a patient a medication comprising a combination of at leastone active therapeutic agent and a marker which was not chemically partof the active therapeutic agent itself, but which was detectable ingaseous exhaled breath; obtaining a sample of the patient's gaseousexhaled breath; analyzing the sample of the patient's breath utilizingan electronic nose to detect the marker in gaseous exhaled breath toascertain the presence or absence of the marker in the patient's breath.The presence of the marker being taken as an indication that the patienttook the medication at a prescribed time and in a prescribed dosage andthe absence of the marker being taken as an indication that the patientdid not take the medication at all or at a prescribed time or in aprescribed dosage.

In 2007, another generic application was filed for a MedicationAdherence Monitoring System, published as US 2010-0255598 which is stillpending. That filing is directed generically to inclusion of anon-ordinary isotope (e.g., deuterium) in the marker used in the methodessentially as disclosed and claimed in U.S. Pat. No. 7,820,108.

Further, in 2011, a new patent filing, published in 2013 asWO2013/040494, generically disclosed solid oral dosage forms (SODFs) foruse in combination with the SMART® system.

As will be apparent from a review of this entire disclosure, the presentdisclosure provides a plethora of select improvements, either inspecific components of the SMART® system, as in the device, thecompositions of matter for use in combination with the device inparticular contexts, in methods of making the device or composition ofmatter, or, in combination, to the system as a whole.

By contrast, for example, Proteus Biomedical, Inc., (now known asProteus Digital Health) has taken the approach to medication adherencemonitoring, as disclosed, for example, in U.S. Pat. No. 8,258,962, a“Multi-mode communication ingestible event markers (IEMs) and systems,and methods of using the same”, in which an integrated circuitcomprising a conductive communication module is ingested to confirmmedication adherence by sending out a signal (e.g., an RFID signal) oncethe circuitry has been ingested by a subject with a medication bearingthat circuitry.

AiCure, by contrast, is an artificial intelligence company whichutilizes facial recognition and motion-sensing technology to monitormedication ingestion using a smartphone camera.

These approaches are, of course, distinguishable from metabolic studiesdesigned to determine the functional (phenotypic) efficiency of specificenzyme systems (e.g., CYP 1A2, CYP 3A4) in which metabolism of acompound is determined by including in the compound (a substrate for aspecific enzyme) to be studied a radioactive or non-radioactive butnon-ordinary isotope, as in Katzman, U.S. Pat. No. 5,962,335, since inthat instance, there is no doubt about whether a medication has beentaken, and, in addition, an isotopic label in the active therapeuticagent is required as opposed to a label in a marker included with anactive therapeutic agent.

Likewise, the medication adherence monitoring technology describedherein is distinguishable from, for example, the implantation of a drugdelivery device, such as, for example the osmostic delivery devicedisclosed in Ayer, U.S. Pat. No. 6,283,953, comprising an implantablereservoir having at least one opening for delivering a beneficial agentcontained within an interior of the reservoir to an organ of an animal,an osmotic engine adapted to cause the release of the beneficial agentcontained within the reservoir to the animal, and means fornoninvasively measuring the release of the beneficial agent from thereservoir from outside, of tissue in which the delivery device isimplanted. The Ayer system requires the invasive implantation of amechanical medication delivery device. Noninvasive monitoring isconducted to ensure correct operation of the implanted device, but onceimplanted, there is no question of medication adherence—if the device isimplanted and is operating as it should, the subject receivesmedication. In addition, the Ayer system is not scalable for a largescale clinical trial, thousands of implantation surgeries would berequired to implant the drug delivery device. By contrast, the systemaccording to the present invention does not require the implantation ofa drug delivery device.

Notwithstanding the significant and incremental developments that haveoccurred in this field, some of which are discussed above, none of theknown systems, devices and methods fully meet the need in the art for anintegrated system capable of providing both acute and chronic medicationadherence monitoring options. The present invention meets this need byproviding improved MAMS components, including an optimized SMART®device, improved compositions of matter and methods of making and usethereof, and, in particular, an integrated system in which thesecomponents operate together to accommodate a wide range of medicationadherence monitoring requirements in varying contexts. The presentdisclosure, therefore, represents a quantum leap forward in that anintegrated system is provided herein wherein commercial embodiments of aSMART® device are disclosed in combination with selected embodiments ofSMART® compositions of matter and methods of using such embodiments inoptimized combinations with each other to provide a gold-standard in thefield of acute and chronic medication adherence monitoring.

3.0 SUMMARY OF THE INVENTION

The present invention accommodates a number of aspects of MAMS notheretofore adequately addressed by any known medication adherencemonitoring system. Included in these aspects are improved embodiments ofthe SMART® device, improved embodiments of SMART® composition of matterfor use in combination with the improved SMART® device, and improvedembodiments of methods of making and using the SMART® device andcomposition of matter as an integrated system to address differentcontexts in which medication adherence monitoring is desired. Theseadvances in each of the related elements of the system may be summarizedas follows:

THE SMART ® SYSTEM ACCORDING TO THIS INVENTION COMPRISESCOMBINATIONS/PERMUTATIONS OF THE: SMART ® DEVICE: See section 6 + 8;examples 1-4; FIGS. 1-18 (a) biometric capture concurrent with breathcollection; (b) portable GC with 5 ppb sensitivity for select VOCs; (c)catalytic incineration + IR detection; (d) EBM measurement withoutseparation (e) small footprint device; (f) combinations of (a)-(e);SMART ® COMPOSITION OF MATTER: See Section 7 + 8; examples 5-26; FIGS.19-74 (a) optimized gelatin capsules containing optimized adherenceenabling marker (AEM) formulations with appropriate release kinetics andretention characteristics/barriers to loss/admixture with API; (b)i-AEMs - AEMs containing non-ordinary isotopes which appear as i-EBMs inexhaled breath with appropriate release kinetics and retentioncharacteristics; (c) optimized compositions and methods for surfacecoating of APIs with the AEM; SMART ® METHOD OF MAKING: See Sections6-9; examples 1-26 The SMART ® device; The SMART ® compositions ofmatter; SMART ® METHOD OF USING AND SMART SYSTEM: See Section 8 + 9,FIGS. 1-74; examples 1-28 Embodiments of the device in combination withembodiments of the compositions of matter to achieve definitivemedication adherence monitoring system to enable a method for: (a) AcuteMedication Adherence Monitoring (AMAM) = immediately to about an hour ortwo after a medication is taken; (b) Intermediate Medication AdherenceMonitoring (IMAM) = immediately to 12-24 hours after a medication istaken; (c) Chronic Medication Adherence Monitoring (CMAM) = immediatelyto about 3 hours to greater than 2-3 days after a medication is takenand insight into adherence across multiple doses.

Accordingly, it is a first object of this invention to provide animproved SMART® medication adherence monitoring system.

A further object of the invention is to provide an improved SMART®device.

A further object of the invention is to provide an improved SMART®composition of matter.

A yet further object of the invention is to provide an improved methodof making and using the SMART® system, device, and composition ofmatter.

A further object of the invention is to provide a system for medicationadherence monitoring which enables acute medication adherence monitoring(AMAM), intermediate medication adherence monitoring (IMAM), and chronicmedication adherence monitoring (CMAM).

Those skilled in the art will further appreciate that the inverse ofmedication adherence monitoring is the detection of drug diversionand/or drug counterfeiting. That is, if a subject is definitivelyconfirmed to be taking their prescribed medication, there cannot be drugdiversion or counterfeiting. Conversely, if a subject is thought to benon-adherent, the system and method according to this invention providesa basis for exploration of whether the subject has been prescribed acounterfeit medication or if the subject is diverting their medicationto another person or persons. Therefore, it is a further aspect of thisinvention to provide an improved method, system, and device fordetection of drug diversion or counterfeiting.

In light of the general disclosure provided herein, including thedetailed exemplary support, and the claims which follow, those skilledin the art will appreciate from a review of this entire disclosure, thatthe invention disclosed herein encompasses a system for medicationadherence monitoring comprising at least the following elements:

A state of the art device or apparatus configured to identify and/orquantitate volatile compounds in a gas sample. The device includes atleast one sensor adapted for identification and/or quantitation of avolatile compound of interest present in the gas sample and at least onecapture device which captures volatile compounds in the gas sample. Thesensor is selected from any of an array of known sensors, including butnot limited to metal oxide sensors (MOS sensors), infrared sensors (IR),Surface Acoustic Wave sensors (SAW sensors) or the like. Combinations ofsuch sensors may be included in the device such that the gas orcomponents of the gas introduced into the device is/are contacted witheach such sensor before being released from the device into theatmosphere. The capture device is selective in that, while it isefficient at capture of volatile compounds, especially volatile organiccompounds, it either does not capture at all or is inefficient in thecapture of moisture, hydrogen, nitrogen, or carbon dioxide, present inthe gas sample. These latter components in the gas sample, therefore,merely flow through the capture device and are vented to the atmosphere.The capture device is selected and adapted to further exhibit theproperty of releasing captured volatile compounds for sensing by the atleast one sensor at a time coordinated in the device to coincide withreadiness of the at least one sensor to be contacted with releasedvolatile compounds that had been captured.

A device which includes these elements, to come within the scope of thepresent invention, further must include at least one or a combinationof:

-   -   a. a catalytic incinerator between the at least one capture        device and the at least one sensor which converts volatile        compounds to carbon dioxide and water prior to contact with the        at least one sensor;    -   b. at least one volatile compound separator between the at least        one capture device and the at least one sensor which separates        volatile compounds released by the capture device prior to        contact with the at least one sensor;    -   c. at least one wireless data transceiver;    -   d. an air scrubber for removal of moisture and volatile organic        compounds present in ambient air to provide a scrubbed air        stream for driving volatile compounds through said apparatus;        and    -   e. a battery.

In a first preferred embodiment of this device, the apparatus is adaptedfor identifying and/or quantitating volatile compounds present in theexhaled breath of a subject. The adaptations for this purpose include,but are not limited to at least one or a combination of:

-   -   f. at least one biometric capture device for concurrent capture        of a biometric specific to a subject when the gas sample is        provided by the subject to the apparatus;    -   g. a mouthpiece for delivery of an exhaled breath sample by a        subject to the apparatus, where the mouthpiece is operatively        coupled with an exhaled breath detection sensor; and    -   h. an actuator, such as a push button or touch sensitive screen        element or the like, on the apparatus, for a subject to actuate        to report adherence in taking or administering a dose of a        medication.

In a further preferred embodiment of this device, the apparatus includesat least two sensors with differential sensitivity to a volatilecompound of interest in the exhaled breath of a subject. Whenappropriately selected and configured, as described herein above, thedifferential sensitivity or selectivity of the at least two sensorsallows information to be derived by manipulation, including bycomparison of signals from each such sensor (e.g., addition of onesignal to the other, subtraction of one signal from the other and thelike) about the presence and optionally the amount of a particularanalyte of interest in the exhaled breath sample.

In another embodiment of this aspect of the invention, the deviceincludes at least one or a combination of:

(i) a catalytic incinerator and an infrared sensor adapted to detectwater or carbon dioxide containing non-ordinary but stable isotopes ofcarbon, oxygen or hydrogen;

(ii) a compound separator such as, in a preferred embodiment, a gaschromatograph that is operatively coupled with an air scrubber thatprovides a scrubbed air stream which is driven through the gaschromatograph by a pump;

(iii) a thermally desorbable concentrator column operating as a volatileorganic compound capture device in intimate association with a heatingelement such that, upon heating of the heating element, capturedvolatile compounds are released from the capture device;

(iv) a wireless data transceiver comprising at least one or acombination of: a WiFi transceiver; a mobile cellular data transceiver;a Bluetooth® transceiver; or the like;

(v) a camera operating as a biometric capture device which captures atleast one still image of the subject at the time that the subjectexhales into a mouthpiece incorporated into and in operative couplingwith the device;

(vi) the battery is a rechargeable battery;

(vii) a microcontroller in operative electrical coupling with othercomponents of the apparatus;

(viii) a limit of detection for a volatile compound of interest of 5-100parts per billion to as low as several parts per trillion. Preferably 10ppt-5 ppb.

In another aspect of this invention, the device described above is usedin a method for medication adherence monitoring, which comprisescontacting the device (e.g., breathing into the device; separatelycapturing a breath or breaths in a capture device, (e.g., a breathcapture bag, a breath capture column which efficiently captures organiccompounds in the exhaled breath but which does not efficiently capturemoisture, hydrogen, nitrogen or carbon dioxide), and then releasingcaptured breath or breath components into the device), with an exhaledbreath sample of a subject. In a preferred embodiment of this method,the device is used by a subject in combination with a medication adaptedfor provision of a marker which the device is configured to detect inexhaled breath. Thus, in this embodiment of the method, an ActivePharmaceutical Ingredient (API) is provided with or without a separateAdherence Enabling Marker (AEM). The API, the AEM, or both when taken oradministered to a subject generates a sufficient quantity of an ExhaledBreath Marker (EBM) in the exhaled breath of the subject to be detectedby the at least one sensor. In a preferred embodiment, the device isused to detect the EBM within a specified time period after a subjecttakes or is administered or applies a single dose of the medication. Ina preferred embodiment according to this aspect of the invention, thedevice and the medication are selected and configured such that the EBMis detectable in the exhaled breath of the subject after the subjecttakes or is administered or applies multiple doses of the medication,and/or in relatively wide windows of time, or even random times, after asubject has or should have taken one or multiple doses of a medication.As described herein above and as further supported by specific examplesprovided herein below, the medication formulation options and devicefeature options are sufficiently malleable that the method can bepracticed in any or each of these modes to reliably achieve AMAM, IMAM,CMAM, as needed for a given medication, subject, or set of clinicaltrial requirements.

The method described herein may, in one preferred embodiment, bepracticed with an API, an AEM, or both, which includes a non-ordinaryisotope. As described herein, the non-ordinary isotope is preferablyselected to exist in the API, AEM or both such that the non-ordinaryisotope is included in a resulting EBM, when it appears in the exhaledbreath of a subject that takes or applies or is administered such amedication. Preferably, the non-ordinary isotope appears in the exhaledbreath of a subject at a known and/or predictable concentration in theexhaled breath of such a subject at a time after taking such amedication which is convenient, or randomly selected, for the subject toprovide an exhaled breath sample to the device. Accordingly, the methodaccording to this aspect of the invention includes embodiments in which:

(a) A SMART® (Self Monitoring And Reporting Therapeutic) medication isprovided to a subject which enables monitoring of the subject'sadherence in taking or administration of at least one ActivePharmaceutical Ingredient (API) included in the medication in which themedication includes: (i) an i-API fraction, that is a known percentageof the total amount of the API delivered, which includes at least onenon-ordinary but stable isotope; or (ii) an i-AEM, an Adherence EnablingMarker, which includes at least one non-ordinary but stable isotope; or(iii) both an i-API fraction and an i-AEM; such that, on taking oradministration of the medication by or to the subject, an i-EBM, (anExhaled Breath Marker comprising at least one non-ordinary but stableisotope), is produced in the exhaled breath of the subject; and/or

(b) An i-EBM is detected and/or quantitated in the exhaled breath of asubject utilizing a device which comprises a component element thatstrips the exhaled breath sample of moisture and carbon dioxide, withoutimpacting (e.g., removing, depleting) the i-EBM. The device usedaccording to this method may further include a catalyst for convertingthe i-EBM to carbon dioxide and water, such that: (a) the isotope fromthe i-EBM is included in the water fraction, such that, followingcatalysis, isotopically labeled water is quantitated in the exhaledbreath sample; and/or (b) the isotope from the i-EBM is included in thecarbon dioxide fraction, such that, following catalysis, isotopicallylabeled carbon dioxide is quantitated in the exhaled breath sample.

The system according to this invention includes a medication comprisingan API and an AEM, wherein the AEM is contained in a chemical form orwithin a barrier adequate to contain loss of the AEM and/or to preventthe AEM from contacting the API prior to being taken or administered bya subject. In a preferred embodiment, the chemical form or barrierfacilitates rapid release of the AEM and/or API in a subject to permitmedication adherence monitoring by measurement of an EBM in the exhaledbreath of a subject within a specified time period, either immediatelyor a short period (up to about an hour), or a longer period, (from aboutone hour up to and including several days) after a medication isingested by, taken by, is administered to or applied onto the subject.In a medication for use according to the method or in the systemaccording to this invention, the barrier in a preferred embodimentcomprises a softgel capsule shell which is optionally coated by abarrier, surface coating, or materials which prevent loss of the AEMfrom the capsule. Alternatively, or in addition, the AEM is provided ina chemical form that is stable until exposed to the biologicalenvironment of the subject, whereupon it quickly forms the AEM in situand is then expired in the exhaled breath as the EBM. In a furtherpreferred embodiment of such a medication, the AEM comprises either orboth (a) a non-ordinary isotope; (b) butanol, isopropanol, or both,either or both of which may include a non-ordinary isotope, or otherselected secondary alcohols, or other AEMs. In a further embodiment, themedication includes a surface coating comprising an i-AEM. Given thesensitivity of a D₂O detector described herein, a low quantity (e.g.,1-10 mg) of a deuterated AEM placed on the surface (partial surface ortotal surface) of SODFs (solid tablets, capsules) is adequate to permitmedication adherence monitoring. Surface coating and containment, forexample, in a blister pack or equivalent preserves the AEM or i-AEM onthe surface of the SODF. Likewise, in some embodiments, the AEM isincorporated into the surface coating of the SODF so that it does notrequire storage in a blister pack, but rather can be stored in astandard pill bottle.

In a further aspect of this invention, the Adherence Enabling Marker(AEM) composition comprises at least one of:

(a) at least one secondary alcohol which when ingested produces anExhaled Breath Marker (EBM) detectable in the exhaled breath;

(b) at least one flavorant to mask taste reactions associated with theAEM following ingestion of the AEM composition; and

(c) at least one bulking agent or other functional excipient to permitreliable filling of softgel capsules and stable storage of the AEMcomposition within a softgel capsule.

In iterations of this embodiment of the AEM formulation for medicationadherence monitoring, the AEM formulation includes permutations orcombinations of the following: the AEM is preferably a secondaryalcohol, e.g., 2-butanol, isopropyl alcohol, or both, or othercombinations and equivalents of other AEMs as disclosed herein; thebulking agent comprises PEG-400, or any of a wide array of other bulkingagents known in the art (fractionated coconut oil; Acconon®surfactant/dispersing agents, e.g., MC-8-2; Phosal® lipids; oleic acid(refined); various grades of PEG; HPC, e.g., Klucel®; povidone; Capmul®emulsifiers; potassium acesulfame); the flavorant, if present, comprisese.g., vanillin, DL-menthol, or both, or other flavorants known in theart. In specific AEM compositions according to this invention, theformulation consists of: (a) 20 mg 2-butanol+0.7 mg DL-menthol+5 mgvanillin+9.3 mg PEG-400; or (b) 40 mg 2-butanol+1.4 mg DL-menthol+10 mgvanillin+18.6 mg PEG-400; (c) 20 mg of 2-butanol alone; (d) 40 mg of2-butanol alone; (e) combinations of 2-butanol and isopropyl alcohol,alone or in combination with other excipients. Of course, those skilledin the art will appreciate that the amount of AEM used may be varied,depending on the concentration of EBM required to be detected in theexhaled breath. This may require as little as 1 μg and as much as 200mg. It is generally sufficient to utilize between about 1 mg and 50 mgof, e.g. 2 butanol to measure butanone increases in the ppb range in theexhaled breath. The advantage of combinations of AEMs is that the SMART®device according to this invention can detect either or both AEMs in theexhaled breath, and either or both EBMs generated from the AEMs (e.g.,butanone and acetone), and any interferents can thereby be identified ifthe ratio of AEMs/EBMs is inconsistent with a detected compound whichcould not have been generated from the AEM in the relative amountdetected in exhaled breath.

In light of the many optional configurations described herein for thedevice, medication, and method according to this invention, the systemfor medication adherence monitoring according to this inventioncomprises the use of an apparatus as described herein in combinationwith a medication comprising an API, an AEM, or an API and AEM, whereinthe API, the AEM, or both are present in a chemical form or containedwithin barriers adequate to contain the API, the AEM, or both from lossor contact between the AEM (if present) and the API. In such a system,it is preferred for the barrier to facilitate rapid release of the AEM,the API or both, in a subject to permit medication adherence monitoringby measurement of an EBM in the exhaled breath of such a subjectgenerated from the AEM, from the API, or both, within a specified timeperiod after the medication is ingested or otherwise administered orapplied to or by the subject.

In further embodiments according to this aspect of the invention, thesystem includes:

(a) a SMART® drug comprising an API, an AEM, or both which generate amarker or markers, Exhaled Drug Ingestion Marker(s) (EDIMs) thatappear(s) in the exhaled breath of humans or other vertebrates, toconfirm definitive medication adherence, and

(b) a SMART® device, which accurately measures the EDIMs and optionallyprovides medication reminder functions, and orchestrates criticaladherence information flow between the relevant stakeholders; whereinthe SMART® drug comprises an Adherence Enabling Marker (AEM) compositioncomprising: (i) at least one secondary alcohol which when ingested orotherwise taken or administered to a subject produces an Exhaled DrugIngestion Marker (EDIM) detectable in the exhaled breath of the subject;(ii) an adequate quantity of flavorant such that greater than 90% ofrecipients of the AEM composition report little or no adverse tastefollowing ingestion of the AEM composition; and (iii) an adequatequantity of bulking agent to permit reliable filling of soft-gelcapsules and stable storage of the AEM composition within a soft-gelcapsule. Preferably, the SMART® device accurately measures the EDIMs,optionally provides medication reminder functions, and orchestratescritical adherence information flow between the relevant stakeholders.This is achieved at least in part by selecting a sensor from the groupconsisting of miniaturized Gas Chromatography linked to any or acombination of a Metal Oxide Sensor (mGC-MOS), a surface acoustic wave(SAW) sensor, an infrared (IR) sensor, and an ion mobility spectroscopy(IMS) sensor.

Those skilled in the art reading this disclosure will further appreciatethat the present invention provides a method for using an AdherenceEnabling Marker, AEM_(x), (which may include use of an API acting as itsown marker), or measuring an Exhaled Drug Ingestion Marker X, EDIM_(x)produced on ingestion of an AEM_(x) comprising characterizing thepharmacokinetics, including concentration-time relationships ofappearance and clearance of EDIM_(x) in the exhaled breath of a subject.In a further refinement of this method, AEM_(x) comprises a non-ordinaryisotope of an atom which constitutes AEM_(x) such that the non-ordinaryisotope is included in EDIM_(x) in the exhaled breath upon dosing of asubject with a medication comprising AEM_(x). In a particularlypreferred embodiment, the non-ordinary isotope is deuterium. Where anon-ordinary isotope is included in the EBM, because the backgroundlevel of deuterated molecules in the exhaled breath is essentially zero,the Limit of Detection, LOD, of the method is only constrained by thelowest concentration of EDIM_(x) that the sensor used in the method isable to reliably measure, thereby providing a lookback period limitedonly by the LOD of the sensor, and the relationship of steady stateconcentration EDIM_(x) (related to its half life) and the mass of theAEM delivered to the subject. This method may also be practiced with acombination of AEMs and EDIMs concurrently, (that is AEM_(x), EDIM_(x);AEM_(A), EDIM_(A); AEM_(B), EDIM_(A); AEM_(C), EDIM_(C); AEM_(N),EDIM_(N)). See Example 28 herein below for detailed description of thisaspect of the invention.

An optimized device or system according to this invention is optimizedby including in the device:

-   A. a sensor selected for accurate detection in the exhaled breath of    at least one subject of at least one Exhaled Drug Ingestion Marker    X, EDIM_(x) produced on ingestion of at least one Adherence Enabling    Marker, AEM_(x);-   B. data storage (as in hard drive, flash drive, EEPROM, in a form    now known or which is developed in the future) operatively coupled    to the sensor, for retention of data generated by the sensor in the    course of characterizing the pharmacokinetics of the EDIM_(x) in the    exhaled breath of a subject, Y, or in a population of subjects, Z;    and-   C. computing means, (including, for example, a programmed central    processing unit) which compares each such measurement for each    subject or population of subjects with stored data, as described    herein below, for said subject or population of subjects, preferably    in real time or near real time. For each measurement of the    concentration of EDIMx, a measure of adherence A is generated by the    computing means for each subject.

The characterizing data for storage preferably includes measurementdata, to within defined confidence limits, of:

-   -   a. the Limit of Detection (LoD) of a sensor included in said        device for said marker;    -   b. the background level of said marker or interferents in said        subject or population of subjects;    -   c. the half life of appearance (t_(1/2a)) and elimination        (t_(1/2a)) of said marker from the exhaled breath of said        subject or population of subjects;    -   d. the steady state (“SS”) concentration of said marker in the        exhaled breath at various time points during Adherence Enabling        Marker (AEM) dosing, selected from the group consisting of        trough (C_(Trough,SS)) maximum (C_(MAX,SS)), and other time        point post dosing of the AEM concentrations of said subject or        population of subjects; and    -   e. the time required to attain the maximum concentration        (T_(MAX)) of said marker from the exhaled breath of said subject        or population of subjects.

Such a device according to this invention is preferably configured tointegrate the pharmacokinetic parameters defined above to provide anadherence lookback window, T_(AdhWindow), defined as the period of timerequired for the marker (EDIM) concentration in breath of the subject todecay from an initial value (C_(EDIMo)) to a lower concentration(C_(EDIM,Limit)):

$T_{AdhWindow} = {\frac{t_{{1/2}e}}{0.693}*{\ln \left( \frac{C_{EDIMo}}{C_{EDIMLimit}} \right)}}$

wherein:

C_(EDIMo)=original or starting concentration of marker (EDIM) in breathat times equal to or greater than T_(MAX) (i.e., C_(EDIMo)≦C_(MAX)) ofsaid patient;

C_(EDIMLimit)=the final concentration of EDIM in breath of said patient,provided that, if C_(EDIMLimit) denotes the limit of EDIM detection dueto the device LoD or background interference, it would define themaximum T_(AdhWindow); and t_(1/2e)=the elimination half life for saidEDIM.

Such a device preferably exhibits a T_(AdhWindow) between about 1 hourand about 400 hours, and includes a sensor with a LoD for the marker ofbetween 1 part per trillion and 5 parts per billion or, naturally,higher, as the higher the concentration the easier it is to define asensor with an adequate LOD. In one preferred embodiment, the sensor isadapted to distinguish between ordinary and non-ordinary isotopespresent in EDIMs and volatile compounds which otherwise would interferewith selective measurement of EDIMs in the exhaled breath.

The invention disclosed herein includes an improved system formedication adherence monitoring wherein the system comprises:

-   -   (A) an Adherence Enabling Marker (AEM) which is administered to        or is taken by a subject concurrently with or substantially        concurrently with a medication according to a medication dosage        regimen the adherence to which by the subject is to be        monitored. When the subject is adherent to the medication        regimen, the AEM or a metabolite of the AEM, referred to as an        Exhaled Drug Ingestion Marker (EDIM), is detectable in the        exhaled breath of the subject over a time period T following        each dose of the medication being taken;    -   (B) a device adapted for (i) sampling, collection, or both        sampling and collection of exhaled breath or a portion of        exhaled breath of a subject, and (ii) detection, measurement or        both detection and measurement of the EDIM, (which can be the        AEM or a metabolite of the AEM) if present in the exhaled breath        or portion of exhaled breath of the subject; and (iii) a        display, data output, or both, reporting the detection,        measurement or both of the EDIM.

The improvements in such a system as disclosed herein comprise at leastone or a combination of the following elements, with respect to the AEM,the device or both:

-   -   (a) the AEM is characterized such that the kinetics of        appearance and clearance of the EDIM in the exhaled breath of a        subject or a population of subjects is sufficient to provide        known confidence limits for such kinetics to be valid for a        given subject, such that an optimal time for detecting the EDIM        in the exhaled breath of a subject over time period T is not        restricted to a time associated with only a single dose of        medication;    -   (b) the AEM is selected for use in combination with a device        adapted for medication adherence monitoring of a subject by        detection of an EDIM, such that an incremental change in the        EDIM is detected in the exhaled breath of the subject each time        a medication dose containing the AEM is taken by or is        administered to the subject;    -   (c) the device comprises a means for distinguishing and/or        separating volatile compounds present in the exhaled breath of a        subject and a detector for detecting, measuring or both        detecting and measuring such volatile compounds or derivatives        of such compounds (e.g. D₂O), wherein the device further        includes at least one of the following elements:    -   i. means for subject biometric capture and reporting for        definitive identification of a subject concurrent with the        subject providing an exhaled breath sample via a mouthpiece. In        this embodiment, the mouthpiece and subject biometric capture        device are configured to enable reliable identification of the        subject each time a breath sample is provided by the subject;    -   ii. a breath flow sensor;    -   iii. a wireless data transceiver;    -   iv. a breath collection and sampling subsystem operatively        coupled with the mouthpiece;    -   v. an air scrubber;    -   vi. a rechargeable battery pack subsystem; and    -   vii. a microcontroller subsystem in operative electrical        coupling with between one and all electrical components of        elements (i)-(v).

To support development and facilitate regulatory filings, a number ofcomplementary in vitro (benchtop) and clinical (human) studies have beencarried out to characterize the SMART® Adherence System. In terms ofhuman exposure, the system has been safely used to date in 32 humanstudies (oral, sublingual, and microbicide administration routes),encompassing 1,293 experiments in 303 subjects and 8,474 breathanalyses. Of particular note, three recent prospective, blinded,randomized, cross over clinical validation studies (127 subjects with472 experiments and 2,464 breath analyses) using the SMART® AdherenceSystem designed for oral medications were executed that focused onidentifying an optimal adherence-enabling marker (AEM) formulation andcarrying out receiver operating characteristic (ROC) curve analyses tomake an optimal cutoff determination and assess diagnostic performance.System performance was favorable across a wide range of subject factors,including age, gender, race, body mass index (BMI), disease conditions,and time of food ingestion, and even in populations enriched withsubjects who chronically consumed alcohol and/or used tobacco products.Specifically, after ingestion of the SMART® Adh Caps containing anoptimized AEM formulation, the following notable clinical study (study1, see examples) outcomes were found: 1) greater than 98% of subjectsgave an overall positive response (detection of breath marker by theSMART® Device), and 2) adherence accuracies exceeding 95% are achievedwhen a 20-90 min breath marker detection window is employed. We disclosemethods, compositions and devices for extending that windowconsiderably, over many hours to days, and/or over more than one dose ofmedication. Given the above results, we conclude that the SMART®Adherence System holds significant promise as a novel technology todefinitively measure and monitor medication adherence in variousclinical settings.

Based on the extensive disclosure provided herein, other objects,advantages, and permutations, variations, combinations or equivalents ofthis invention will be clear to those skilled in the art from a reviewof the complete disclosure and appended claims.

4.0 BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Provides an illustrative example of how the system and methodaccording to this invention works. The figure illustrates enzymaticcatalysis and resultant exhalation of 2-butanone following oralingestion of 2-butanol (40 mg) in subjects (n=7). Panel A: metabolism ofthe AEM, 2-butanol, by on-alcohol dehydrogenase (ADH) to generate thevolatile product, 2-butanone, an Exhaled Drug Ingestion Marker(EDIM)/Exhaled Breath Marker (EBM). Panel B: breath concentration-timerelationship for the exhalation of 2-butanone (an EDIM) in breathfollowing consumption of 2-butanol at time 0 min. Data shown aremean±SD. *, P<0.05 for a given time point compared to time point 0 min.The arrow denotes time of ingestion of a capsule containing 2-butanol.Concentrations less than the level of 1.0 parts-per-billion (ppb) arenoted as “<LOD”. As can be seen from this figure, 2-butanol as the AEMand 2-butanone as the EBM provides the ability to measure adherence overa time period of a few minutes to about one hour or so from the time oftaking a medication containing the AEM.

FIGS. 2A and 2B. Graphic representations of a Handheld Miniature SMART®Device according to this invention.

FIG. 3. SMART® device block diagram showing breath sampling, separation,biometric capture, data and instruction display, data communication,microcontroller and power subsystems.

FIG. 4A-E. SMART® GC Subsystem Interconnect Block Diagrams.

FIG. 5. Graphic representation of a first embodiment of a Mouthpiece(Straw).

FIG. 6. Technical Drawing of Disposable Mouthpiece.

FIG. 7A-C. Mouthpiece Sensor, Breath Flow Sensor and Vapor Inlet in twodifferent embodiments of the SMART® device according to this invention.

FIG. 8. Flow Diagram for Breath Collection and component separation in aminiature GC (mGC) embodiment of the SMART® device according to thisinvention.

FIG. 9. Exemplary representation of a SMART® mGC chromatographicseparation of isoprene, acetone, and 2-butanone in human breath.

FIGS. 10A and 10B. Photograph of internal architecture of one exemplaryembodiment of internal components of the SMART® device.

FIG. 11. Photograph of internal architecture of obverse view shown inFIG. 10 in one exemplary embodiment of the SMART® device.

FIG. 12. Air flow path for scrubbed carrier air in the SMART® device.

FIG. 13. Exemplary representation of one embodiment of a user interfaceand SMART® device operational flow diagram.

FIG. 14. SMART® device logic flow diagram.

FIG. 15. SMART® device logic and data flow diagram.

FIG. 16a-g . A Prospective Randomized Cross Over Clinical Study in 50Subjects to Determine the Optimal Configuration of the SMART® AdherenceSystem: Effect of Four Adherence-Enabling Marker Formulations andValidation of the SMART® device operation; 16 a Age; 16 b Gender; 16 cEthnicity; 16 d Body Mass Index (BMI); 16 e Time From Last Meal; 16 fAlcohol Use; 16 g Tobacco Use; none of these factors appeared to beconfounding factors.

FIG. 17a -j. 2-Butanone Breath Concentration-Time Relationship—Effect ofAdherence-Enabling Marker (AEM) Formulation, see FIG. 17 a; Δ2-Butanone(Change In Concentration From Baseline Values) Breath Concentration-TimeRelationship, see FIG. 17b ; Effect of Adherence-Enabling Marker (AEM)Formulation on Δ2-Butanone Breath Concentration-Time Relationship:Effect of AEM Formulation; Individual Δ2-Butanone Concentration-TimeCurves in 50 Subjects: 20 mg 2-Butanol—See FIG. 17c ; IndividualΔ2-Butanone Concentration-Time Curves in 50 Subjects: 20 mg 2-ButanolCombo see FIG. 17d ; Individual Δ2-Butanone Concentration-Time Curves in50 Subjects: 40 mg 2-Butanol—See FIG. 17e ; Individual Δ2-ButanoneConcentration-Time Curves in 50 Subjects: 40 mg 2-Butanol Combo—SeeFIGS. 17f and 17g ; Distribution of 2-Butanone Concentrations by Time,AEM Formulation, and Concentration Threshold Levels; Percent of Subjects(N=50) with Δ2-Butanone Concentrations 5 PPB; see FIG. 17h ; Percent ofSubjects (N=50) with Δ2-Butanone Concentrations 7.5 PPB—See FIG. 17i ;Percent of Subjects (N=50) with Δ2-Butanone Concentrations 10 PPB—SeeFIG. 17 j.

FIG. 18a-i . Effect of Meal Timing on Δ2-Butanone Concentrations AcrossAEM Formulations—See FIG. 18a ; Covariates: Tobacco and Alcohol Use—SeeFIG. 18b ; ΔT_(Max): Effect of AEM Formulation see FIG. 18c ; CumulativeFrequency (%) of Subjects Achieving ΔT_(Max) by Time and Formulation—SeeFIG. 18d ; ΔC_(Max): Effect of AEM Formulation—See FIG. 18e ; ΔAUC:Effect of AEM Formulation—See FIG. 18f ; SMART® Device Performance: Full2-Butanone Concentration Range—See FIG. 18g , which shows 2-butanonebreath concentration-mGC response relationships by device across fourAEM formulations; relationship between 2-butanone concentration and mGCresponse is curvilinear (i.e., square root function), but is highlylinear in regions, including lower concentrations (0-100 ppb; see FIG.18h ) and higher (300-3000 ppb) concentrations relevant to the doses of2-butanol ingested; Sensitivity of mGC SMART® Devices: Low 2-ButanoneConcentrations=0-100 ppb; see FIG. 18h ; stability of a softgelcontaining the AEM according to this invention is shown in FIG. 18 i.

FIG. 19. Schematic of optional features, permutations and combinationsof features for embodiments of the SMART® device (Type II) according tothis invention.

FIG. 20. Schematic details of a first optional arrangement of Type IISMART® device components.

FIG. 21. Schematic details of a second optional arrangement of Type IISMART® device components and output example from analysis of i-EBM.

FIG. 22. Schema showing the metabolic fate of selected ordinary isotopeand non-ordinary isotope labeled alcohols, aldehydes and carboxylicacids.

FIGS. 23-53. Schemes showing particular biochemical conversions ofselected molecules to exemplify fate of particular atoms which may actas non-ordinary isotopes for use as i-AEMs/i-EBMs in combination with anembodiment of the SMART® device (Type II) according to this invention.

FIG. 54. Breath Concentration-Time Profile from a 30 mg bolus ofisopropyl alcohol (IPA; isopropanol; 2-propanol) delivered in a size 0capsule to a fasting subject, showing IPA induced increase abovebaseline for acetone in the exhaled breath of the subject. See FIG. 55for mGC analysis after ingestion of 10 mg IPA.

FIGS. 55A and 55B. First derivative of the mGC profile for 0, 5, 10, 15,and 30 minutes post ingestion of 10 mg IPA; 55B shows the ratio of firstderivatives for the acetone/isoprene mGC profiles.

FIGS. 56-59. GC/MS and OrbiTrap (LC/MS/MS) Analysis.

FIGS. 60-61. Real time Analysis of Acetone Breath Kinetics followingIngestion of 3 mg d8-Isopropanol Using the OrbiTrap LC/MS.

FIG. 62. Real time Analysis of Acetone Breath Kinetics following tworepeated ingestions of 10 mg d8-Isopropanol and 10 mg Isopropanol Usingthe OrbiTrap LC/MS.

FIG. 63. Breath kinetics of exhaled 2-butanol and 2-butanone followingthe concurrent ingestion of 2-butanol and ethanol; A. Mass spectrum of asingle breath sample taken before the ingestion of 2-butanol withethanol. Of the four analytes high-lighted, only acetone can bepositively identified. The small peak at 90 is likely due to isotopicinterference from the unknown background component appearing at m/z=88and not 2-butanone; B. Mass spectrum of a single breath sample taken 5min after the ingestion of 2-butanol and ethanol. Ethanol, 2-butanoneand acetone are now present as prominent peaks, but 2-butanol is barelydetectable above baseline; C. Breath kinetics of 2-butanone andd6-acetone following ingestion of neat 2-butanol (40 mg) andd8-isopropanol (20 mg) after lunch, baseline breath; D. 5 minutes postingestion; E. 25 minutes post ingestion; F. D6-Acetone was detectable inthe breath one minute after ingestion of the d8-isopropanol. The graphwas generated using data from orbitrap LCMS. The orbitrap was configuredto capture sequential spectra (˜5 spectra per second) and these spectrawere recorded for the duration of the experiment (60-90 min usually) toproduce a real time continuous trace. The electrospray interface on theorbitrap was modified to allow a subject to blow exhaled breath samplesdirectly into the source while the mass spectra were being collected.The rapid clearance of the breath samples from the source allowed us tocapture and characterize mass spectra from exhaled breath samples inreal time. In theory we could use the orbitrap to capture anddistinguish every exhaled breath that a subject makes during anexperiment but in practice we typically don't need to collect more thanone breath sample per minute.

FIG. 64. Breath kinetics of exhaled 2-butanol and 2-butanone followingthe concurrent ingestion of 2-butanol and ethanol; (A) Plotting the peakheight of each compound of interest as a function of time yields thebreath kinetics for each potential breath marker. Even with a reasonabledose of ethanol present in the stomach, the kinetics of 2-butanoneappears unaffected (or at least very similar to a typical responsefollowing the ingestion of just 2-butanol) and no significant 2-butanolwas detected; (B) Breath kinetics of 2-butanone and d6-acetone followingingestion of neat 2-butanol (40 mg) and d8-isopropanol after lunch.

FIG. 65. FTIR Analysis of Acetone and Isopropyl Alcohol along with theirperdeuterated isotopologues; 65A tracing showing the infrared spectrumfrom a NIST Webbook Gas Phase IR Spectrum of 2-Propanol; 65B there isprovided a spectrum obtained by the inventors using a Thermo Nicolet6700 FTIR Gas Phase IR Spectrum of 2-Propanol.

FIG. 66. 66A tracing of the FTIR analysis of acetone and d6-acetoneshowing clear areas where these spectra are distinguishable from eachother; 66A′ shows an expanded portion of the tracing from FIG. 66a inwhich this is very clearly shown; 66B tracing of the FTIR analysis ofIPA and d8-IPA, again showing clear areas where these spectra aredistinguishable from each other. FTIR Spectra A shows the HC═O stretchfor acetone at 2985 cm⁻¹ versus the DC═O stretch for d6-acetone at 2261cm⁻¹. FTIR Spectra B shows the H3C—OH stretch for IPA at 2970 cm⁻¹versus the D3C—OH stretch for d3-IPA at 2231 cm⁻¹. Both of thesespectral shifts are easily distinguishable.

FIG. 67. FTIR Spectra of Acetone and Isopropyl Alcohol with theirperdeuterated isotopologues, with a detail of each tracing in theFingerprint Region (1170 cm⁻¹ to 1300 cm⁻¹, 8.5470 mm to 7.6923 mm).

FIG. 68. FTIR Analysis of Acetone and Isopropyl Alcohol along with theirperdeuterated isotopologues; 68A, FTIR Spectra of d6-acetone versusBlank Breath, with details of portions of these spectra being shown inFIGS. 68B and 68C.

FIG. 69. Breath kinetics of exhaled d6-acetone following topicalapplication of d8-isopropanol in a carbomer gel or oral ingestion ofd8-isopropanol; left axis=100 mg d8-IPA oral; right axis, 20 mg d8-IPAoral and 240 mg d8-IPA topical.

FIG. 70-74. Breath kinetics of exhaled d6-acetone following theingestion of 100 mg of d8-isopropanol per diem for 5 days. FIG. 70 showsthat native acetone peak heights remained reasonably constant throughoutthe study.

FIG. 71 shows that baseline levels for ion 82 (the ion used to monitord6-acetone) were low and less than 1000 (<1% of typical acetone levels).An increase of exhaled d6-acetone was apparent within 2-4 minutes ofingesting each dose of d8-IPA. Maximum breath levels were achieved after1-2 h and ranged from 450,000 to 800,000 peak height (˜2-5×concentrations of endogenous/native acetone).

FIG. 72 shows that 24-hour trough levels were relatively unchanged overthe course of the study and were ˜10% of peak maximum.

FIG. 73 shows that the decline of d6-acetone in exhaled breath followeda first order decay (2-24 h post ingestion). The rate constant (k) forthis decay was consistent throughout the study.

FIG. 74 shows that at this rate of elimination, approximately 6-10% ofmaximum peak response remains after 24 h. Such kinetics should producesteady-state trough levels that are also ˜10% of the maximum peak. Thismatches the observed trough levels during the study.

FIG. 75. 75 a—A sample mGC chromatogram of a human breath samplefollowing ingestion of the hard gel capsule containing 60 mg 2-butanoland 60 mg 2-pentanone; 75 b-concentration-time relationships for humansubjects (n=5) to exhale 2-butanone or 2-pentanone after concurrentlyorally consuming encapsulated 2-butanol (60 mg) and 2-pentanone (60 mg)immediately after time 0 min; six replicates were conducted for eachsubject; note the logarithmic scaling of the ordinate axis; thehorizontal dashed line designates the lower limit of detection (LOD) forthe miniature-gas chromatograph; data shown as mean±standard deviationfor parts-per-billion (ppb) based on molar fractions; the overallconcentration-time plots for 2-butanone and 2-pentanone shown in FIG.75b demonstrate the similarity of these relationships for both exhaledmarkers; 75 c—inter-individual variability of the concentration-timerelationships for human subjects (n=5) to exhale 2-butanone (Panel A) or2-pentanone (Panel B) after orally consuming 2-butanol (60 mg) and2-pentanone (60 mg) immediately after time 0 min; six replicates wereconducted for each subject; note the logarithmic scaling of the ordinateaxis; the horizontal dashed line designates the lower limit of detection(LOD) for the miniature-gas chromatograph; data shown as mean±standarddeviation for parts-per-billion (ppb) based on molar fractions; thelegend applies to both panels; 75 d—intra-individual variability of theconcentration-time relationships for human subjects (n=5) to exhale2-butanone (Panel A) or 2-pentanone (Panel B) after orally consuming2-butanol (60 mg) and 2-pentanone (60 mg) immediately after time 0 min.;the same five subjects composed each replicate; the horizontal dashedline designates the lower limit of detection (LOD) for the miniature-gaschromatograph; data shown as mean±standard deviation forparts-per-billion (ppb) based on molar fractions; the legend applies toboth panels; 75 e-Concentration of 2-butanone compared to that ofconcurrently collected 2-pentanone for all specimens collected (n=240)from human subjects (n=5) after orally consuming 2-butanol (60 mg) and2-pentanone (60 mg); note the regressed solid line with dashed 99%confidence limits; data shown as mean±standard deviation forparts-per-billion (ppb) based on molar fractions.

FIG. 76. Shown in Panel A of FIG. 76 is the 1^(st) derivative mGCresponse (proportional to EDIM breath concentration) in a Type 1 SMARTDevice for acetone and 2-butanone as a function of breath sampling timespost ingestion of the capsule. Shown in Panel B is the same data as adifference from baseline (little change in the appearance of the2-butanone curve due to little or no background, but shifting of theacetone curve downward after subtraction of background acetone).

FIG. 77. IPA as an AEM using a Type I SMART® Device according to thisinvention. 77 a mGC-MOS Chromatograms for IPA Calibration Curve; 77 bIPA Calibraton Curve analyzed on the mGC-MOS; 77 c acetone in exhaledbreath (concentration in ppb) vs. time. In this example, Ingestion of100 mg of isopropyl alcohol (IPA) rapidly increased the acetoneconcentrations in breath above baseline values. The rise was greaterthan 6× (baseline: 450 ppb vs maximum: 2800 ppb) that of baselineacetone concentrations.

FIG. 78: Fundamental pharmacokinetic relationships for six successiveadministrations of an oral drug. The light line is the pattern of drugaccumulation during repeated administration of a drug at an intervalequal to its elimination half life, when drug absorption is very rapidrelative to elimination. The concentration maxima approach 2 and theminima approach 1 during the steady state. The heavy line depicts thepattern during administrating of equivalent dosage by continuousintravenous infusion. Curves are based upon a one compartment model. Thex axis represents time, as indicated by multiples of elimination halflife (t_(1/2a)). Reference: modified FIG. 1-6, page 27, Goodman andGilman, The Pharmacological Basis of Therapeutics, 8^(th) Edition, 1993,Pergamon Press, New York, N.Y. Abbreviation Key: C_(Trough), troughconcentration of EDIM (circle symbols); C_(MAX), maximum concentrationof EDIM in breath (horizontal dotted lines).

FIG. 79. First Dose PK using d8-Isopropyl Alcohol (IPA) as the AEM. Thed6-acetone breath concentration-time data following the 1^(st) oral doseof d8-IPA (100 mg) in a specific subject, as depicted in FIG. 72, isshown. The experimental data was curve fit (parameter estimates±SE) toEquation 1 using a non-linear, least squares (Marquardt-Levenburg)algorithm (SigmaPlot 11, Systat Software, Inc., San Jose, Calif.).According to the curve fit (R²=0.84), during the 1^(st) 24 hr dosingperiod, the values of t_(1/2a) and C_(Trough) level were 8.50 hr and 229ppb, respectively. Note how absorption was faster than metabolism. PerEquation 2, T_(Max) was 1.46 hr. Thus, the F_(Lost) of d6-acetone duringthe 1^(st) dosing interval with a dosing interval of 24 hr, according toEquation 6, is 0.859, and from Equation 5 the accumulation factor (AF)is 1.165. Thus, at steady state QD dosing, the EDIM C_(Trough) (Equation8) and C_(MAX) (Equation 7) levels of d6-acetone are 267 ppb (=1.165×229ppb) and 1819 (=1.165×1561) ppb, respectively. Using a limiting EDIMconcentration of 100 ppt, the T_(AdhWindow) for C_(Trough) and C_(MAX)levels of d6-acetone according to Equation 9 is 96.8 hrs (4.0 days) and120.3 hrs (5.0 days), respectively. Note: the logarithmic scale is usedon the y axis.

FIG. 80: d6-acetone (EDIM) concentration-time curve in a human after 5sequential doses (D1 to D5) of d8-IPA (100 mg) with adherence “lookback” windows shown at various device LoDs. With a steady stated6-acetone C_(Trough) level of 267 ppb and QD dosing (dosing interval=24hr), according to Equation 9, if the sensor LoD was 10 ppt, 100 ppt, and1 ppb we would have adherence “look back” windows of 125.1 hr (5.2 d),96.6 hr (4.0 d), and 68.6 hr (2.9 d), respectively. These times areindicated by the short vertical lines on the time axis. Note: Becausethere is no significant background d6-acetone in breath, the limit inthis situation will be the device LoD. The y axis is plotted on the log10 scale.

FIG. 81: Simulated EDIM concentration-time relationships generated fromEquation 1 following ingestion of d8-IPA (40 mg) and d10-2-butanol (40mg) using actual (experimental) human PK parameters for IPA and2-butanol. The rate constant of 2-butanone, which is immediately andcompletely generated from 2-butanol, for absorption (k_(s)) andelimination (k_(e)) were 0.025/hr and 0.367/hr, respectively. The rateconstants of acetone, which is immediately and completely generated fromIPA, for absorption (k_(a)) and elimination (k_(e)) were 2.40/hr and0.0815/hr, respectively. In the case of 2-butanol administration,between dosing (QD), the trough concentrations always return to baselinevalues. Thus, the presence or absence of d8-2-butanone in breath can beused to effectively detect and prevent deceit by subjects when usingd8-IPA for AMAM and/or CMAM. In other words, because the d8-2-butanonegenerated from d10-2-butanol has a short t_(1/2e), its presence shouldnot be there if a breath is being provided later than 3 hours afteringesting the medication, or if performing a breath sample to measureC_(Trough) for acetone. Hence, it can serve to prevent deception andeliminate potential interferents to the system. For example, in a 2breath scenario with QD morning (8 AM) dosing of a medication containingthe AEMs d8-IPA and d10-2-butanol, unlike d6-acetone, d8-2-butanoneshould not be present in the baseline breath sample during the 8 AMmorning dosing. The lack of 2-butanone in breath ensures that thesubject did not simply ingest the medication containing the AEMsimmediately before the 8 AM dosing when they were randomly called toprovide a breath sample to the SMART® device to ensure compliance.Likewise, if the subjects were randomly called at night to providebreath samples at 8 PM (12 hours after the daily morning dose), again,no d8-2-butanone should be present. The latter approach has theadvantage of providing major convenience to the subjects (one breathscript at night) without having to provide breath samples during thebusy morning time). Note: the logarithmic scale is used on the y axis.

FIG. 82: Procedure to Use d6-Acetone (EDIM) Trough Concentrations(C_(Trough)) to Determine EDIM elimination half life (t_(1/2a)) and theAdherence Look Back Window (T_(AdhWindow)) using C_(Trough) at theIndividual Subject Level. Shown in the top panel is hypothetical acetone(EDIM) concentration-time data, modeled after inputting experimentalvalues into Equation 1, for a specific subject receiving an oralmedication containing 100 mg d8-IPA at a dosage interval of 1 day(administered once per day, or QD) for an introductory 7 day testperiod, which serves to acclimate the subject to the SMART® AdherenceSystem and determine steady state trough levels of d6-acetone. The onlyparameter measured in this subject is C_(Trough) for acetone, asindicated by the circles in the top panel. The C_(Trough) values aremeasured just prior to administering the new dose of medicationcontaining 100 mg d8-IPA. The bottom panel shows the C_(Trough) versustime over the 7 dosing days at one dose per day. The experimentalC_(Trough)-time data was curve fit to the equation shown in the bottompanel using a non-linear, least squares (Marquardt-Levenburg) algorithm(SigmaPlot 11, Systat Software, Inc., San Jose, Calif.) to determine aC_(Trough) at steady state (C_(Trough,SS)) and the elimination rateconstant (k_(e)) of 646 ppb and 1.282/day, respectively. From Equation4, this k_(e) value corresponds to a d6-acetone (EDIM) t_(1/2e)=13.0 hr.Per Equation 9, this value of t_(1/2a) using a Type 2 SMART device(IR-based) with a LoD of either 100 or 10 ppt, correspond to values ofT_(AdhWindow) of 164.6 hrs (6.9 d) and 207.8 hrs (8.7 d), respectively.Note: In Equation 9, the C_(Trough,SS) is now C_(EDIM,o) and the LoD isC_(EDIM,Limit) because with d6-acetone there is no backgroundinterference. If a subject continues to reliably take his/her medicationlinked to the d6-IPA once per day at approximately the same time eachday, the d6-acetone C_(Trough) levels stay constant. In other words, ifa subject is randomly called to provide a breath sample to establish atrough level at the usual time in the morning when the medication isingested, the value of C_(Trough) should be the same (within the rangeof values) as what was determined from the 7 day introductory period. Incontrast, if the C_(Trough) level measured when the subject is randomlycalled is lower than the C_(Trough,SS), the period of time since he/shedid not take their medication can be calculated by using Equation 3. Forexample, during a random check, the EDIM C_(Trough) was found to be 50ppb, far below the expected C_(Trough,SS) value of 646 ppb. How long wasthe subject non-adherent? Using Equation 3, the elapsed time since thelast dose was 48 hrs, or 2 days (=13 hrs/0.693*ln (646 ppb/50 ppb). Thissubject will need to be counseled and may require daily adherenceassessments versus random calling.

FIG. 83: A. Breath acetone normalized to baseline as measured by the mGCfollowing the ingestion of a placebo capsule (dashed line) and a capsulecontaining 100 mg of Na-2-propyl carbonate. B. Breath acetone (dashedline) and 2-butanone (solid line) as measured by the mGC following theingestion of 100 mg of Na-2-butyl-carbonate.

FIG. 84A-D show the ability, using different AEM strategies, to achievedifferent rates of EBM production, from as quick as 10 minutes fromingestion for peak EBM to much longer peak EBM production times.

5.0 DETAILED DISCLOSURE OF THE PREFERRED EMBODIMENTS ACCORDING TO THEINVENTION

It is acknowledged at the outset that this patent disclosure provides ahighly detailed, definite and enabling written description of asophisticated set of technological improvements which in sum and incooperation with each other provides those skilled in the art withreasonable certainty as to which elements to include and whichcombinations of elements are needed to produce a commercially viable,highly flexible, and integrated system for medication adherencemonitoring. As a result, a new definition of the state of the art isprovided by this disclosure. What follows is a road map for negotiatingthis detailed disclosure.

At least in part because of the significant adaptability of the presentsystem to desired medication adherence monitoring modalities, thefollowing conceptual framework is provided at the outset as a guide, ormap, as to how the various cooperating components of the new systeminterface with each other to provide the operative system exhibitingsufficient in-built flexibility to accommodate definitive medicationadherence monitoring in at least the following significantly differentcontexts: Acute, Intermediate and Chronic Medication AdherenceMonitoring (AMAM, IMAM and CMAM, respectively).

In determining how to design, assemble, and optimize a SMART® system toprovide “gold standard” performance for acute medication adherencemonitoring (AMAM), intermediate medication adherence monitoring (IMAM),and/or chronic medication adherence monitoring (CMAM), four key factorsare involved:

1) the half life of the EDIM in humans,

2) the concentration of EDIM or EBM in breath,

3) the sensitivity of the sensor to detect the EDIM or EBM, and

4) the level of background EDIM/EBM interference that may be present inbreath.

The triad of circumstances consisting of an EDIM having the longest halflife in breath being detected with the most sensitive sensor with nobackground interference (e.g an EBM already present or other breathmarkers that could mimic the EBM to the sensor) provides an optimalSMART architecture for a CMAM system.

In contrast, a triad of circumstances consisting of an EDIM having ashort half life in breath being detected with a less sensitive sensorwith significant background interference (EBM already present or otherbreath markers that could mimic the EBM to the sensor) provides a SMARTarchitecture most suitable as an AMAM system. In such a system, it maybe desirable to utilize a baseline breath, (prior to a subject having anAEM introduced into their system, to determine a profile of markers inthe breath). Where it is known that there is little or no interferencepossible, (e.g. utilizing an i-AEM, as described herein below), a singlebreath may be all that is required. In addition, a single AEM may beutilized in each such mode (AMAM, IMAM, CMAM), different AEMs may beused for each such mode, or combinations of AEMS may be utilized toachieve definitive medication adherence monitoring and exclusion ofinterferents.

The ability to use this technology to produce a “look back” on overallmedication adherence, (with or without using the system on a dailybasis), over a preceding time period as disclosed further herein below,is clinically important, novel and inventive. On the one hand, to carryout ideal pharmacometric modeling, the expert wants dose by dosedocumentation and timing between dosing (interdosing intervals). On theother hand, the ability to conveniently monitor medication adherenceover a wide range of time periods, or even at random times in the courseof a medication regimen, substantially and significantly expands themedication adherence options available, beyond those of any knownsystem, for definitive medication adherence monitoring.

5.1 Acute Medication Adherence Monitoring (AMAM):

In this context, medication adherence is monitored typically on adose-to-dose basis, and usually from immediately or almost immediately(seconds to minutes) after a given dose of a medication is or shouldhave been taken, up to about an hour after a given dose has been orshould have been taken. In the art to date, this is the typical contextfor medication adherence monitoring. That notwithstanding, as will beapparent from a review of the complete disclosure which follows, thepresent invention disclosure provides novel and inventive advancesrelevant to the SMART® medication adherence device, compositions ofmatter, methods of making and using these and an integrated system forSMART® medication adherence monitoring. The time frame for monitoringmedication adherence per this aspect of the invention is typically fromas immediately as possible after a medication is taken by a subject upto about an hour or two after the medication is taken or administered inwhich a marker according to this invention is included with themedication for appearance and detection in the exhaled breath. Foroptimal marker absorption and expression of the Exhaled Breath Marker(EBM) in the shortest amount of time possible, it is desirable for AMAMenabling markers (AEMs) to have significant gastric absorption, whilefor IMAM and CMAM this is less critical (i.e., the marker may be takenup in the duodenum, or lower in the digestive tract). We have found thatisopropyl alcohol (IPA) is an excellent marker for both AMAM, IMAM andeven CMAM, as it is gastrically processed but also rapidly generated anEDIM (i.e., acetone) after oral ingestion that has a longer half life inbreath—see further discussion herein below—than does butanol.

5.2 Intermediate Medication Adherence Monitoring (IMAM):

In this context, medication adherence is monitored typically on a morethan single dose-to-dose basis or, even if just on a dose-to-dose basis,the time-window for monitoring is substantially more flexible thanhaving to confirm adherence within an hour to two hours after amedication is taken. That is, a major advance provided by the presentdisclosure is that it enables medication adherence monitoring to occurimmediately (if significant gastric absorption of the AEM occurs) to aperiod of several hours (up to a day) after a given dose of a medicationis or should have been taken. In this embodiment the system has featuresof AMAM (pill by pill adherence) and IMAM (adherence look back window upto one day). In contrast, if the AEM is not significantly absorbed inthe stomach and generates EDIMs with longer half lives in breath, thesystem could be used to monitor IMAM but not AMAM. In the art to date,there is no known system which can provide definitive medicationadherence monitoring with the flexibility of this much delay from thetime of taking a medication to the time when adherence has to beconfirmed. The time frame for monitoring medication adherence per thisaspect of the invention is typically from about one hour to up to abouttwelve hours following a given medication dose in which a marker isincluded with the medication for appearance and detection in the exhaledbreath. Thus, monitoring according to this aspect of the invention maybe conducted five, six, seven, eight, nine, ten, eleven or even twelvehours after a given medication dose is taken. That is, there isincreased flexibility such that adherence may be confirmed any timeduring a specified window after taking a dose, at pre-specified timepoints within the relevant window, or at random times within the window.

In addition, as will be apparent from a review of the completedisclosure provided herein, more than one dose of a given medication maybe confirmed in such time period, and doses of different medications maylikewise be monitored in this time frame.

5.3 Chronic Medication Adherence Monitoring (CMAM):

In this context, medication adherence is monitored typically on a morethan single dose-to-dose basis, and the time window for medicationadherence monitoring post dose is even further extended. Insight intowhether a subject is following a medication regimen as instructed isobtained. That is, a major advance provided by the present disclosure isthat it enables medication adherence monitoring to occur at any time,including many hours or even days after a given dose of a medication isor should have been taken. In the art to date, there is no known systemwhich can provide definitive medication adherence monitoring with theflexibility of this much delay from the time of taking a medication tothe time when adherence has to be confirmed. The time frame formonitoring medication adherence per this aspect of the invention istypically from about eight hours, and up to about forty eight hours ormore following a given medication dose in which a marker is includedwith the medication for appearance and detection in the exhaled breath.Thus, monitoring according to this aspect of the invention may beconducted eight, nine, ten, eleven, twelve, twenty four, forty eight oreven more hours after a given medication dose is taken. In addition, aswill be apparent from a review of the complete disclosure providedherein, more than one dose of a given medication may be confirmed insuch time period, and doses of different medications may likewise bemonitored in this time frame.

5.4 Layout and Contents of this Patent Disclosure

In the disclosure which follows, we take up, in turn, detailed andenabling written description of:

In Section 6—the SMART® device according to this invention is describedin detail, with particular emphasis on improvements made therein overand above the known generic description of such a device, withparticular focus on improvements in the device for purposes of enablingAMAM, IMAM, and CMAM;

In Section 7—the SMART® composition of matter and methods of making anduse thereof is/are described in detail, with particular emphasis onimprovements made therein over and above the known generic descriptionof such a composition of matter, with particular focus on improvementsin the composition for purposes of enabling AMAM, IMAM, and CMAM;

In Section 8—with reference to the SMART® device according to thisinvention and the SMART® composition of matter, we next take up adetailed description of the improved SMART® system and methods of makingand use thereof, with particular focus on improvements in the system forpurposes of enabling AMAM, IMAM, and CMAM.

In Section 9—specific but non-limiting exemplary support is provided toextend the enabling written description and to provide guidance onspecific implementations of the invention in different contexts.

Various permutations and combinations of these aspects of the inventionenable the practice of the AMAM, IMAM, and CMAM configurations of theinvention mentioned herein above. To practice this invention, an“Adherence Enabling Marker” or “AEM” is included in a medication dosagewhich results in the production in exhaled breath of an “Exhaled DrugIngestion Marker” or “EDIM”, also referred to herein as an ExhaledBreath Marker or “EBM”. The AEM and EDIM may be the same compound, orthe EDIM may be a metabolite of the AEM. Table 1 below provides aconvenient guide to some of the key permutations and combinations asdisclosed and described in detail in the written description whichfollows:

TABLE 1 SMART ® Composition and Device Combinations Optimized for AMAM,IMAM, CMAM Exemplary SMART ® SMART ® Device SMART ® Device CompositionEmbodiment; Embodiment; SMART ® Embodiment - ordinary non-ordinary ModeAEM EDIM/EBM isotopes isotopes AMAM Short half Ketone, GC-MOS GC-IR ±life - e.g., e.g., 2- catalytic secondary butanone, incinerationalcohols 2- (e.g., 2- pentanone) butanol, 2- pentanol) ± nonordinaryisotopes IMAM Longer half Ketones, GC-MOS GC-IR ± life - e.g., e.g.,catalytic isopropyl acetone incineration alcohol ± non- ordinaryisotopes CMAM Longer half ketones, GC-MOS GC-IR ± life - e.g., e.g.,catalytic isopropyl acetone, incineration alcohol, more ketones complexfrom alcohols (2- larger heptanol; alcohols; cyclohexanol), sulfidessulfur from containing sulfur food additives containing (e.g., fooddimethyl additives disulfoxide, allicin) ± nonordinary isotopes

According to one embodiment of the SMARM device according to thisinvention, and methods of using the device and compositions of matter,the use of non-ordinary isotopes in combination with catalyticincineration is described in detail herein below. Those skilled in theart will appreciate from this summary disclosure and the detaileddisclosure which follows, that in practicing the present invention,there is the need to consider the interplay of at least the followingparameters:

Practice of this invention for IMAM or CMAM involves the use of AEMswith longer half lives in the biological system, including in theexhaled breath, than those used for AMAM. The longer the half life ofthe AEM, the longer the potential “lookback” period the AEM enables.There are also mass of marker considerations relevant to practicing thisaspect of the invention. The greater the mass of marker present, thegreater the potential lookback period. Maximizing the “lookback” period,however, also depends on the amount of background and noise presentwhich can confound accurate measurement of the EDIM in exhaled breath.Use of non-ordinary isotopes in the AEM which are retained in the EDIMgoes a great distance, as disclosed in detail herein below, to extendingthe “lookback” period and minimizing signal noise. As will becomeapparent, all of these considerations require optimization for a givendosage form, medication, and adherence regimen. The guidance providedherein teaches those skilled in the art to utilize appropriate markerswith selected half-lives, masses, device/detector embodiments and dosageforms accommodating different marker delivery options in combinationwith each other in optimized configurations to facilitate definitivemedication adherence monitoring in the AMAM, IMAM, and CMAM contexts towhich the present system is adapted.

6.0 IMPROVED SMART® DEVICE AND METHODS OF MAKING AND USE THEREOF

From U.S. Pat. No. 7,820,108, for a “Marker detection method andapparatus to monitor drug compliance”, a device is generically disclosedto determine whether a patient has taken a medication which operates byproviding to a patient a medication comprising a combination of at leastone active therapeutic agent and a marker which was not chemically partof the active therapeutic agent itself, but which was detectable ingaseous exhaled breath; obtaining a sample of the patient's gaseousexhaled breath; analyzing the sample of the patient's breath utilizingan electronic nose to detect the marker in gaseous exhaled breath toascertain the presence or absence of the marker in the patient's breath.The presence of the marker being taken as an indication that the patienttook the medication at a prescribed time and in a prescribed dosage andthe absence of the marker being taken as an indication that the patientdid not take the medication at all or at a prescribed time or in aprescribed dosage. That, in essence, defines the basis of the SMART®device, composition of matter, method and system known in the art.

Per the present disclosure, as will be seen from the detaileddescription provided herein below, the art is significantly advanced bygreatly refining and extending what has been possible to date. Thisincludes the establishment of a SMART® device which has a detectionlimit as low as 5 parts per billion (ppb) for particular EDIMs (e.g.,2-butanone, using 2-butanol as the AEM). Detection at these and lowerconcentrations (see below) are established for this device withconfidence limits of at least 90% and higher (see the examples). Wherenon-ordinary isotopes are utilized as part of the marker, detectionlimits in the parts per trillion (e.g. 10 PPT-1000 PPT; or 10 PPT-1 PPB;or 100 PPT-10 PPB) are enabled by particular embodiments of the SMART®device described herein. Improved combinations of biometric captureconcurrent with sample collection are provided to ensure definitivemedication adherence monitoring and elimination or substantially reducedpossibility for “gaming the system”. Portability, reliability and otherenhancements are likewise provided.

In general, the device of the present invention may take any one of thefollowing forms, each of which is described in detail herein below:

Exhaled Device Exhaled Exhaled Exhaled Breath Type Breath Breath BreathCompound Desig- Compound Compound Compound Incineration nationConcentration Separation Detection then Detection I + + + − II + +--------> + III + − + −

Each of these device types and their mode of manufacture and operationis taken up in turn herein below, following which, specific compositions(including AEMs) for use with a given device type are described and thensystems integrated for use of a given device type in combination with agiven composition are described.

6.1 Detailed Description of a First Embodiment (Type I) of the ImprovedSmart® Device:

A SMART® device according to this embodiment of the invention is adevice comprising integrated subsystems for reliable and accuratemedication adherence monitoring when a SMART® medication is taken by oris administered to a subject. The device at the heart of this inventionis, where compound separation occurs, a miniature Gas Chromatograph(mGC) integrated with a sensor, such as a Metal Oxide Sensor (MOS), oran Infrared Sensor, or a Surface Acoustic Wave (SAW) sensor, togetherreferred to herein as the mGC-MOS, mGC-IR, or mGC-SAW, respectively. Thedevice provides integrated exhaled breath collection, analysis,biometric capture for subject identification, alarms, and datacommunications capabilities.

A Self Monitoring and Reporting Therapeutics (SMART®) apparatusaccording to this embodiment of the invention facilitates definitivedocumentation of medication adherence, as described herein below.

In a preferred embodiment, the SMART® system uses FDA-approved foodadditives, termed adherence-enabling markers (AEMs), which are or whichgenerate volatile compounds, which appear in the exhaled breath,including the AEM itself or metabolites thereof in vivo, referred toherein as the Exhaled Drug Ingestion Marker, or EDIM, or Exhaled DrugEmplacement Marker, or EDEM, to distinguish between ingested medicationswith a marker (EDIM) and medications that are delivered non-orally,e.g., vaginally, rectally, transdermally, etc. (the EDEM). EDIMs andEDEMs are collectively referred to herein as Exhaled Breath Markers,EBMs. The EDIM or EDEM is exhaled by a subject following ingestion,emplacement or other means of administration of a medication includingthe AEM. Measurement of these markers and/or metabolites thereof in abreath sample unambiguously documents adherence (ingestion,administration or application of the medication). Where the AEMs are FDAdesignated Generally Recognized as Safe (GRAS) compounds, they areco-packaged or co-formulated with an active drug, also referred toherein as the Active Pharmaceutical Ingredient (API), into a capsule,tablet, cream, suppository, transdermal patch, or any other appropriatedosage form, in a manner that preferably alters neither the drug'smanufacturing processes nor bioavailability. Of course, the AEM may justas well be associated with a placebo, active control or other clinicalmaterial, rather than the API, and the same or different AEM's may beused to tag different API's, placebos and/or active controls. Onceingested or otherwise administered, the AEM(s) is/are absorbed by thestomach and small intestine, or is taken up across the skin, vaginal orrectal lining, and which then appears directly in the exhaled breath oris metabolized to a volatile marker(s) which appear(s) in exhaled breath(see FIG. 1) according to kinetics known for that marker. Theconcentration(s) of the EBM(s) in a breath sample (˜20 mL) isautomatically measured by a portable, lightweight, miniature gaschromatograph (mGC) or other compound separation technology included inthe SMART® device with minimal subject effort. For the first time, tothe best of the knowledge of the inventors of this device, as furtherdescribed herein below, a portable gas chromatographic apparatus isprovided which, in combination with a sensor (e.g., a MOS sensor, anInfrared sensor, a SAW sensor, or the like), provides low parts perbillion or even parts per trillion sensitivity with precision andaccuracy, for particular analytes in exhaled breath. Thus, for example,using 2-butanol as the AEM, the EDIM, 2-butanone, is measured by thedevice according to this invention in the exhaled breath of subjectswith confidence at as low as 5 ppb within fifteen to twenty minutes ofingestion of the AEM. See the examples below for details, which showthat the device according to this invention provides highly linearresponses at low 2-butanone concentrations (0-100 ppb), which arerelevant to yes/no AMAM adherence decisions (rise in concentration=5-10ppb).

By measuring the metabolite(s) in breath, one can be assured that thesubject did, indeed, consume or otherwise receive the medication becausenative gastric wall and hepatic enzymes (e.g., metabolism of secondaryalcohols by αα-alcohol dehydrogenase) are needed to metabolize theAEM(s) to the volatile, exhaled metabolite(s), i.e. the EDIM. Similarly,for the EDEMs, once in the biological system, appropriate uptake isdemonstrated by appearance of EDEM(s) in the subject's breath. All data(date/time stamps, breath chromatographs, yes/no adherence assessments,mGC self-diagnostic quality assurance logs) are stored locally in themGC device on an, e.g., internal USB flash drive or equivalent storagemedium for later collection and/or transmitted in near real-time usingintegrated encrypted Health Insurance Portability and Accountability Act(HIPAA)-compliant wireless or cellular router technology to a centraldata repository for analysis.

Additional, optional, data streams are available to investigators orother clinical study personnel should the study or medication regimenrequirements warrant collection when compared to subject privacyconcerns: 1) a camera in the SMART® device is time-gated to concurrentbreath collection; this biometric capture (e.g., facial picture; in oneembodiment, if the biometric data captured does not match biometric datastored in the device or in a central data collection facility, thebreath collection may be terminated, or the data may be flagged, orappropriate personnel may be alerted) allows investigators todefinitively confirm that the breath analyzed by the SMART® deviceoriginated from a specific subject at a particular time (when the breathsample was collected), and, 2) the concentration of other compounds,e.g., ethanol in a subject's breath sample that may be of particularinterest to investigators in a given field (e.g., for investigatorsstudying psychotropic drugs, or drugs with CNS effects, it is relevantto know if observed behavioral effects arise as a result of the studymedication or due to confounding effects produced by ingestion of othercompounds, such as ethanol). These data can likewise be stored locallyon the SMART® device and/or transmitted to a HIPAA-compliant datarepository.

Those skilled in the art will appreciate that in place of or in additionto the camera, other biometric or subject identification means may beemployed. For example, rather than a camera, a retinal scanner may beused. Alternatively, each subject may be accorded a radio frequencyidentification (RFID) transmitter or the like, so that actuation of theSMART® device includes confirmation by the device that the RFID of thesubject providing a given exhaled breath sample is the appropriateindividual being monitored. In yet another alternative, the device isadapted to detect an RFID on a blister pack, medication container or themedication itself to confirm appropriate medication and/or dose is beingtaken. Of course, unless implanted, intentional “gaming” of the systemcould potentially still occur by, for example, handoff of an RFID tag bya given subject. Accordingly, biometric confirmation concurrent withexhaled breath sample provision is preferred.

Data acquired by the device are logged into secure, for exampleinternet-based, HIPAA-compliant storage for review by authorizedinvestigators anywhere on the globe with an internet or equivalentdistributed data connection. Investigators may choose to actively reviewthe data on a daily basis to understand day-to-day adherence (activemanagement), to maintain data securely in a blinded fashion untilassignment unmasking (passive management), or some combination ofactive/passive review desired by the study team. Considerableflexibility may be built into this aspect of the system. For example,Data may or may not be reviewed as it is acquired. If reviewed, it maybe reviewed in a blinded or unblinded context (with respect to subjectidentity, treatment modality), and action can be taken based on incomingdata review or not. In a preferred embodiment according to thisinvention, biometrics are encrypted. In a further preferred embodiment,the biometric data are automatically checked against a biometric recordof a given subject, without the need for any human access to thebiometric. In yet a further preferred embodiment according to thisinvention, photographic images of a subject are obtained via a cameraadjusted for focus to a very close focal length, so that essentiallyonly the face of the subject is captured in the image, without much orany background capture, to avoid privacy concerns. As the camera is timegated to breath sample provision, other privacy concerns are likewiseeliminated.

These data allow researchers to know if subjects were actuallyingesting/administering the assigned research article (e.g., aparticular study medication), and following scheduled dosing. Thisinformation is important when assessing the safety and efficacy of adrug. As a result, dose-to-dose intervals andpharmacokinetic/pharmacometric/pharmacodynamic drug modeling options areavailable from this system to inform ongoing treatment modalities. Thehealth outcomes associated with suboptimal adherence to a drug could beassessed, and motivations for adherence in different states (e.g.,healthy/ill; home/travelling) could be investigated since adherence databy time/date is made available by this system. In addition, this systemenables reliable study of the effects of behavioral interventions toimprove adherence. Clinical investigators will likely identify other newuses for this system as it becomes available for full use in a broadswath of studies across multiple populations and locations.

The key to understanding adherence, like any scientific data, ismeasuring it. The breath-based SMART® technology system provides thistool to scientists and clinical trial investigators.

From a subject's perspective, the adherence measurement system is easilyportable and designed to be self-administered by subjects in their ownresidences, workplaces, or in an appropriate clinical setting. Thisfeature offers significant subject convenience and investigator economicbenefits compared to frequent appointments with study staff for directlyobserved therapy (DOT), the “gold standard” of adherence, (to the extentthat up to now any gold standard could be said to exist). Additionally,no study staff is required for daily assessments, the SMART® systemprovides a cost-effective option for definitive adherence monitoring anddata acquisition, as compared with DOT, which is generally availableonly during business hours and not during weekends or holidays. Overall,the change in subject behavior is simply an approximately 5 or so secondbreath exhalation into the mGC within an optimal time period afterorally consuming or otherwise emplacing a medication comprising a SMART®AEM. A somewhat longer lag time may be required for transdermallydelivered medications, but the principles are the same. By altering AEMdose and/or type, the rate of appearance in breath and duration ofmarker persistence in breath can be adjusted to maximize versatility ofthe SMART® system. All breath analyses and data logging/transmissionsare preferably automatic (i.e. do not require subject action).Alternatively, in one embodiment according to this invention, the deviceis adapted to receive an active indication by a subject that a dose ofmedication has been taken, and that data may be included in the acquireddata that is logged, transmitted and available for analysis. Usabilitystudies conducted under NIMH 2R44MH081767-02A1 with an early prototypeof the SMART® device indicated a high degree of satisfaction with thissystem by HIV/AIDS patients receiving adherence measurement for highlyactive antiretroviral therapy (HAART) (Morey 2012 J. Clin. Pharm; MoreyAIDS beh. 2012; van der Straten AIDS Beh. 2013).

6.1.1 SMART® Medication Adherence

To date, Xhale, Inc. has focused its development efforts on commercialdevelopment of the SMART® adherence devices for use in combination withSolid Oral Dosage Forms, SODFs, particularly tablet- or capsule-basedmedications, which are swallowed, enter the stomach, and are absorbed inthe gastrointestinal tract. In this case, definitive adherence isindicated within minutes or at most hours from the time of ingestion ofsuch a SODF by the detection in the exhaled breath of a metabolite of anAEM, also referred to herein as a taggant (preferably a GRAS flavorantand most preferably a direct food additive) which may also be the EDIMor which is the source for the production of the EDIM. The taggant ispackaged together with the API in the final SODF, although means forseparation of the taggant from the API is preferably employed, accordingto the disclosure found in WO2013/040494. In such embodiments, theSMART® system has successfully employed 1) various formulationstrategies that incorporate taggants into the final dosage form,preferably without or minimally altering the CMC per se of the CTM(Clinical Trial Material), investigational drug, or marketed drug, and2) a mGC-MOS as the SMART® device to measure the EDIMs.

Prior to describing elements of the current invention in detail, a briefreview of some key aspects of taggant chemistry is provided here.

Consider a scenario where a patient with a specific disease ingests anactive drug, A, for treatment, which is metabolized by enzyme(s) to Alplus other irrelevant metabolites. In this example, a safe taggant(e.g., GRAS flavorant) without pharmacological activity called T, whichmay be metabolized to a major metabolite, T1 plus other irrelevantmetabolites, is packaged with A. Thus, the two relevant metabolicreactions are: 1: A->A1+others 2: T->T1+others.

With regard to measuring a marker that appears in breath, the EDIM(s),which can be measured to verify that A was orally ingested by thepatient, four chemical candidates are available: 1) A; 2) a majormetabolite of A, Al; 3) a taggant, T, which was ingested with themedication containing A; or 4) a metabolite of any taggant (T), T1,which was generated via enzyme metabolism of a taggant (T). Theappearance of T1 about 5-10 minutes later in the breath can be used todocument the active drug A (the Active Pharmaceutical Ingredient, or APIor CTM) was actually ingested. To optimize performance of the adherencesystem, we have developed novel compositions of matter (see Section 7below) wherein a taggant is included in, for example, a soft gel capsuleor in another physical or chemical form which is stable, (see exemplarysupport for e.g. a carbonate which is surface coated onto or surfaceprinted onto an API dosage form, while preserving, where considerednecessary, an impermeable physico-chemical barrier between the taggantand the API, and which is rapidly converted into the Exhaled BreathMarker, EBM, on introduction into the biological system), and which iswell tolerated by subjects, which generates markers in the exhaledbreath which are quickly and reliably detected, and which do notinterfere with co-delivered APIs.

Any appropriate AEM composition (and resultant EBMs, including EDIMs,EDEMs), including but not limited to the taggants, markers, dosage formsand the like disclosed in, for example, “Marker detection method andapparatus to monitor drug compliance”, U.S. Pat. No. 7,820,108; US2005/0233459; “System and Method of Monitoring Health Using ExhaledBreath”, US2007016785; “Methods and Systems for Preventing Diversion ofPrescription Drugs”, US20080059226; “Medication Adherence MonitoringSystem”, US 2010/0255598; or in WO2013/040494, published 21 Mar. 2013,entitled “SMART™ SOLID ORAL DOSAGE FORMS”, may be used in combinationwith the SMART® device disclosed herein.

6.1.2 the SMART® Adherence Device According to this Embodiment of theInvention

DEFINITIONS AND PRODUCT NAME REFERENCES

Components described as being “operatively coupled” are components thatare at least in communication with each other and operation of one ofthe operatively coupled components has an impact on the operation of theother operatively coupled components. This can include one of theoperatively coupled components directly or indirectly controlling theoperation of the other component, as in a CPU programmed to controlperipheral elements of a device or system. This can also mean thatoperation of the first operatively coupled component results inmodification of the operation of the second component, including whenthe first component does not directly or indirectly control operation ofthe second component.

Contacting a device with a gas means that a sample of the gas isintroduced into the device's operative mechanism for analysis ofcomponents of the gas. This may include separation of components of thegas. It may include detection of particular species in the gas. It mayinclude quantitation of species in the gase. It may include contactingof the gas with sensors of different specificity such that by comparingwhat is sensed by a first sensor with what is sensed by a second sensor,the difference in what the two sensors detect provides affirmativeinformation about the presence, absence and even concentration of agiven gas species.

ACRONYMS

CPU: Central Processing Unit

GC Gas Chromatograph

GUI: Graphical User Interface

lbs: pounds

LPM: Liters per minute

ml: milliliters

mm: millimeters

ppb(v): parts per billion (by volume)

ppm(v): parts per million (by volume)

ppt(v): parts per trillion (by volume)

sccm: standard cubic centimeters per minute

SOP: Standard Operating Procedure

USP: United States Pharmacopoeia

VOC: Volatile Organic Compound

PRODUCT NAME REFERENCES

Throughout the development of the SMART® mGC system, we have utilizedseveral reference names for the device.

-   -   SMARM Model 100 Adherence Monitor    -   SMARM adherence monitor    -   SMARM device    -   Mini GC    -   Handheld Miniature GC    -   Handheld GC    -   mGC    -   mGC-MOS

We also refer to the disposable patient interface as:

-   -   Straw    -   Mouthpiece    -   Disposable straw    -   Disposable Mouthpiece

When used herein, the terms “operative communication” or “operativecoupling” or “operative electrical coupling” mean, based on the contextof where these terms are used, that the described elements communicatewith each other or one element is controlled by another, eitherelectrically or mechanically, based on system design features and/orprogramming scripts included in a controller device to which otherdevices are linked.

EDIM—Exhaled Drug Ingestion Marker, an Exhaled Breath Marker (EBM)generated when an AEM is ingested.

EDEM—Exhaled Drug Emplacement Marker, an Exhaled Breath Marker (EBM)generated when an AEM is applied topically or introduced by a meansother than ingestion.

AEM—Adherence Enabling Marker, which itself can be the EBM (EDIM orEDEM), or which gives rise to the EDIM or EDEM via metabolism, in vivo,of the AEM; while specific secondary alcohols are provided as examples,such examples should be considered non-limiting for the AEM; preferably,the AEM according to this invention is a GRAS compound, including butnot limited to food additives which give rise to volatile metabolites inthe body when metabolized.

EBM—Exhaled Breath Marker (e.g., EDIM, EDEM).

To the extent possible, the same numbering is used for like elementsshown in various representations of the device according to thisinvention, with, not necessarily, all elements being shown in every suchrepresentation.

With reference now to FIG. 2A, there is provided a first representationof one embodiment of the SMART® mGC system 100 according to thisinvention. The SMART® mGC system 100 is an easy-to-use, handheldinstrument which is essentially a miniature gas chromatograph (“mGC”)comprising a housing 110, a display 120 which may also include, in apreferred embodiment, a photographic image capture device 121 toconcurrently document the image of the subject exhaling into the device,an exhaled breath receiving mouthpiece 130, inert to VOCs in the exhaledbreath, also referred to herein as a “straw”, which is inserted into themouthpiece receiver port 131, and an activation or “Start” button 140.Those skilled in the art will appreciate that while this representationprovides a first configuration of the physical parameters of oneembodiment of the SMART® device according to this invention, alternateconfigurations come within the scope of this invention, as shown anddiscussed in the “Alternate Configurations” section of this disclosure.

In FIG. 2B, there is shown an embodiment of the SMART® device, 100,identical to that shown in FIG. 2a , (and therefore elements labeled inFIG. 2a are not again labeled in this figure), with an addedrepresentation of a loudspeaker 141 which provides audible alerts anduser prompts. In this representation, the mouthpiece 130 has beenremoved to more clearly reveal the mouthpiece receiver port 131. On therear panel of the housing 110, there is provided, in appropriateembodiments, an input power jack and electrical power connection 142 forpowering the device or, in an embodiment which includes an internal orexternal rechargeable power pack, recharging the battery pack via anexternal wall transformer. In alternate embodiments, the battery packitself may be exchanged out of the device or be rechargeable or otherforms of replaceable power may be utilized, such as standard disposablebatteries.

The SMART® mGC System according to this invention is designed to analyzegaseous samples (e.g., human breath or breath of other vertebrates) forsuitable organic molecules of clinical interest, and, particularly,EDIMs and/or EDEMs.

Gas chromatography is an extensively used analytical technology, and thephysicochemical basis of its operation is well documented andunderstood. While the principles of operation for the breath analysis(or gas sample analysis) performed by the SMART® mGC are similar to theprinciples of operation for a standard gas sample analysis usingcurrently marketed bench-top gas chromatographs, the specifics of themGC SMART® device according to this invention are unique. Thus, anaspect of the present invention is the provision of a robust,miniaturized, portable, accurate, HIPAA-compliant commercial device andsystems and methods for using this device for medication adherencemonitoring. Naturally, of course, the mGC according to this inventionmay be utilized in a wide variety of applications wherever an accurateportable gas chromatograph would be of use. Thus, for in-the-fieldmonitoring of volatiles, e.g., in the industrial workplace, or tomonitor emissions, the mGC according to this invention would be anaccurate and valuable tool. Naturally, for such applications, featuresincluded in the present disclosure need not necessarily be included—suchas, for example, the biometrics capture discussed herein above.

6.1.3 Device Subsystem Block Diagram

The block diagrams in FIGS. 3 and 4 detail key subsystems and theirinterconnections within the SMART® mGC apparatus according to thisaspect of the invention.

Referring now to FIG. 3, there is shown an embodiment of the mouthpiecethat accepts the breath sample, also referred to as a disposable straw,130, which is configured to supply breath components to a breathdetection and sampling subsystem, 132, which is operatively coupled to agas chromatograph analyzer subsystem 150. On insertion into the device,the mouthpiece 130 is detected by a straw/mouthpiece sensor 133 toconfirm proper engagement and readiness to receive an exhaled breathsample. An ambient air stream is routed via a disposable air scrubber(see description below, FIG. 4C, elements 300-310), to provide a carrierair system for the gas chromatograph analyzer subsystem 150. Amicrocontroller subsystem 160 integrates with the gas chromatographanalyzer subsystem 150, and concurrently controls the operation of acamera and display subsystem 170, and a WiFi, cellular or othercommunication means including data transceiver or mobile cellular datahotspot subsystem 180. A wall power transformer 190 provides power tothe device including, optionally, a rechargeable battery pack subsystem191.

6.1.4 Device Subsystems

Further detail of each subsystem and the order of operative flow of theSMART® device 100 is shown in FIG. 4A, with detailed descriptionprovided for each subsystem being provided in FIGS. 4B-4E.

6.1.5 Mouthpiece Subsystem

In FIG. 4B, the disposable mouthpiece subsystem 130 is shown.Preferably, included in this subsystem is a vent, 136, such that exhaledair passing through the mouthpiece is vented to the exterior of thedevice. Also included in this subsystem are a breath flow sensor 132 toindicate to the system that a breath sample is being received by thedevice 100, and a straw sensor 133, which is activated when a breathcollection straw is inserted into the device 100 for breath samplecollection. Finally, a conduit 134 provides for a metered quantity ofbreath to be routed from the disposable mouthpiece 130 into the SMART®device 100 for gas chromatographic analysis. The breath volume collectedis controlled by the time that the sample pump is energized. The samplerate is controlled by the vacuum pressure developed by the vacuum pumpand the flow resistance presented by the concentrator.

Referring now to FIG. 5, detailed photographs are shown of thedisposable, single patient use mouthpiece (straw) 130 provided tofacilitate collection of the breath sample. FIG. 5A shows themouthpiece/straw from a top view, while FIG. 5B shows the straw bottomview. As can be seen, each straw 130 includes a breath inlet end 135, aflow restrictor/vent port 136, (in this embodiment, the second end ofthe straw is sealed), a breath sample port 137 which couples with theconduit 134 which provides for a metered quantity of breath to be routedfrom the disposable mouthpiece 130 into the SMARM device 100 for gaschromatographic analysis. Finally, there is provided a flow sensor port138 which couples with the breath flow sensor 132. In addition todirectly receiving exhaled breath samples from a subject as the subjectexhales, the SMARM device may also receive samples via gas-sampling bagsor gas-tight syringes by coupling these devices to the breath inlet end135 of a straw, or directly to the breath sample conduit 134.

FIG. 6 provides a schematic showing a first embodiment of how themouthpiece/straw 130 aligns with the device. Failure to align themouthpiece correctly prevents it from locking into place in the SMART®device straw holder 131, particularly with respect to alignment of thebreath sample port 137 and the flow sensor port 138.

FIG. 7A provides a photographic representation of an embodiment of themouthpiece receiver 131 of the SMARM device, including the vapor inletport 134, the breath flow sensor 132, the straw optosensor 133, all ofwhich align with and engage the mouthpiece shown in FIGS. 5 and 6. Alsoshown is the start button, 176.

In a preferred embodiment according to this invention, themouthpiece/straw 130 is simplified to use of a simple tube, as shown inFIG. 2A, open at both ends, 135 and 136 for delivering exhaled breathfrom the subject to the device. In this embodiment, the ports, sensorsand other elements shown in FIGS. 4B, 5, 6, and 7A, are all removed fromthe mouthpiece straw 130 into the docking port, 131, visible from theexterior of the device only as a port, as can be seen in FIG. 2B. Thissubstantially reduces the complexity and cost of the straw andsimplifies the use thereof for the user of the SMART® device. In FIGS.7B and 7C, the internal structure of the straw/mouthpiece port 131 isshown via a cross section through the top of the device 100 through theport 131, represented in FIGS. 2A and 2B. An isolated view of thiscross-section through the port 131 is provided in FIG. 7C. A simplestraw with an inlet and an outlet and no other features other than itbeing inert and of dimensions to tightly fit the port is inserted intothe straw/mouthpiece receiver port 131. In one preferred embodiment, theport comprises a first cylindrical chamber area 139 with a diametersufficient to easily accommodate insertion of the straw 130therethrough. A second area 143 follows area 139 with a diameter whichtapers from that of antechamber 139, which is greater than that of themouthpiece tube/straw, down to a final diameter of a narrowercylindrical area 149, the diameter of which is less than that of themouthpiece tube/straw. The ends of the mouthpiece straw and the surfaceat the start of the cylindrical area 143 are preferably machined to havemating surfaces and taper such that the inserted end of the straw locksin place within area 143 on correct insertion of the end of themouthpiece straw. The inserted end of the straw 130 thus mates with butcannot enter into area 143 much beyond the very initial section of area143 as the narrowing taper thereof prevents this. As a result, anair-tight seal is formed between the external surface of straw 130 andthe internal walls of the mouthpiece receiver port 131 in area 143.Alternative embodiments include providing threading on the ends of thestraw and mating threads in area 143. Further alternative embodimentsinclude press-fit, flanging or other means for the straw end to beretained in the receiver port in an air-tight fashion. In each suchembodiment, exhaled air is channeled from the end of the straw into area143, and from there passes into area 149 and excess exhaled air isvented out of vent port 144. Vent port 144 is in communication with theexternal aspect of the device 100 housing via external vent 145, whichpermits excess exhaled breath and any breath condensate to be dischargedfrom the device. Exhaled air sample port 134 (leading to exhaled breathsample conduit 147 and from there into the separation and detectionsubunits, see below) and flow sensor 132 are both in fluid communicationwith the exhaled air stream by being open to the conduit defined byareas 143 and 149. Correct placement of the straw in docking port 131 isconfirmed by the straw sensor (e.g., an optosensor) 148 shown in FIG.7C. A retaining screw 146 is provided to retain the docking port 131 incorrect placement within device 100. In a preferred embodiment, theinlet port is composed of a material which prevents condensation.Silico-steel, for example, is a preferred embodiment for this element.In a further preferred embodiment, the inlet tube is heated to preventcondensation—particularly important for embodiments of the deviceintended for use in cold climates.

6.1.6 GC Subsystem and Sensor

Referring back to FIG. 4C, detail is provided for the gas chromatographsubsystem 150. Included in this subsystem are the following components:an exhaled breath sample receiver port 151 coupled to the conduit 134,via conduit 147 (preferably which provides the breath sample from thedisposable mouthpiece 130 when a subject exhales into the SMART® device100, as described in detail above. The exhaled breath sample is directedfrom the exhaled breath sample receiver port 151 into a thermallydesorbable concentrator subsystem 200, comprising a hydrophobicconcentrator column 201 around which is wound or otherwise intimatelyassociated a heating coil 202 or equivalent heating element such as athermoelectric heating element, such as but not limited to a Peltierdevice which, when activated, heats the thermally desorbableconcentrator column 201, to thereby desorb any bound compounds from theconcentrator column. A fan 205 is provided to ensure even heatdistribution over the column and efficient and rapid dissipation of heatwithin the enclosure 110. At either end of the thermally desorbableconcentrator column 201, valves, 203 and 204, are provided on theproximal and distal ends, respectively. The valve 203 on the endproximal to sample receiver port 151 controls the receipt of the exhaledbreath sample from the exhaled breath sample receiver port 151 into thethermally desorbable concentrator column 201 when the breath flow sensor132 indicates that an exhaled breath is being received. When theappropriate quantity of exhaled breath sample has been received into thethermally desorbable concentrator column 201, the sample pump isde-energized to stop the collection of the breath sample onto thethermally desorbable concentrator column 201, and any excess air isvented via the vent 330. When the SMART® device 100 is ready to analyzethe breath sample, the heating element 202 heats the concentrator 201 torelease bound compounds, and the valve 203 on the proximate end of theconcentrator 201 opens to permit delivery of bound molecules to the gaschromatograph column 152, housed inside a column oven 153 which includesa heater 154 and temperature sensor 155 for precise regulation of thegas chromatograph column 152. The desorbed molecules travel from theconcentrator 201 via valve 203 through connector 156 and into the gaschromatograph column 152 via GC inlet port 157. At the distal end of theconcentrator 201, valve 204 opens to permit delivery of carrier gas fromthe carrier pump 304 via carrier pump coupling 305, flow restrictor 307,disposable dessicant cartridge 308, port 309 to port 310, and, via valve302 to valve 204 to drive the desorbed molecules into and through the GCcolumn 152. Once the desorbed sample has been delivered, valve 302remains open permitting scrubbed ambient air which has been drawnthrough a disposable charcoal filter 303 to drive the sample through theGC column 152 then through the GC detector 158 and out of vent 159. Aswill be appreciated from this disclosure, coordination of valves 203,204, and 302 is required to ensure that desorbed molecules from theconcentrator 201 are driven into the GC column 152 at the appropriaterate, temperature and pressure. This coordination is achieved by theelectronic microcontroller subsystem, 160, which, in a preferredembodiment, also coordinates the taking of a biometric record, in apreferred embodiment, a photograph, of the subject at the time ofdelivery of the exhaled breath sample.

To ensure that appropriate carrier air pressures are not exceeded, thereis provided a carrier air pressure sensor 311 which feeds back to thecarrier pump 304 via electronic microcontroller 160 to control carrierair pressure. As the desorbed molecules travel from the concentrator 201into the GC column 152 they are fractionated and then detected by a GCdetector 158 and then vented from the SMART® device 100 via vent 159.

Depending on the nature of the molecules to be detected, and theadherence environment in which the device is utilized, the detector,158, may be a MOS detector, an infrared detector, and, as discussed insome detail below, for certain embodiments according to this invention,the detector includes a catalytic incineration feature. While apreferred embodiment according to this invention utilizes a mGC, coupledto a MOS, those skilled in the art will appreciate that other means ofseparation and/or detection may be utilized for a particularapplication. For example, a concentrator and an array of surfaceacoustic wave (SAW) sensors may be utilized as an “electronic nose” inplace of the GC column and MOS sensor.

The chromatographic separation of the various breath components andmarkers occurs on the column 152 which, in one embodiment, consists of a5 meter long piece of 0.53 mm ID metal tubing whose walls are coatedwith a polymeric stationary phase (e.g, Restek MXT BAC-1). Thestationary phase adsorbs and desorbs the various chemical vaporsinjected in the initial plug. The adsorption and desorption rates ofeach vapor vary, depending on physicochemical characteristics such asboiling point and hydrogen bonding affinity. Since a constant stream(nominally 3 sccm) of clean, dry air carrier gas is flowing through thewall-coated metal column, those compounds that are more volatile areswept through most rapidly and emerge from the GC tube 152 at an earliertime than those molecules that are heavier and less volatile. Thedetector 158 that produces a signal proportional to the number oforganic molecules exiting the tube is used to record when the differentmolecules emerge. Thus, each compound can be identified by its retentiontime, and the concentration can be determined by the peak height, whencomparing it to analytical standards of known concentration. The GCdetector used in the SMART® GC is, in one preferred embodiment, asolid-state, metal oxide semiconductor (MOS) chip sensitive to thepresence of oxidizable hydrocarbons.

To provide consistent performance, the SMART® column 152 is operated ata constant temperature, e.g., 40° C. via regulation by the column oven153, and the associated temperature sensor 155 and heater 154. Thetemperature is regulated to keep the temperature steady. Those skilledin the art will appreciate that, and will know from their own skills andfrom the guidance provided herein that, different column packing,temperature, mobile phase and the like are required to optimizeseparation of different components, as needed, of exhaled breath tooptimize detection and non-interference with detection of the EBM.

6.1.7 Stand-Alone Mouthpiece, Camera, Sample Collection Module

In an alternate embodiment according to this invention, the biometric,e.g., time-stamped photograph of the subject, and collection of theexhaled breath sample, are provided as a separate module from theremainder of the apparatus. On provision of the time-stamped breathsample to the remainder of the apparatus, the sample is analyzed as in afully-integrated embodiment. The advantage of this embodiment is thatthe breath sample and biometric may be trapped at any location, withoutthe need to carry the entire device. This creates an even more portableoption for users of the system. The components of this embodiment wouldinclude the breath straw, a camera, a pressure sensor, and a desorbableconcentrator column—as discussed above. On combining this module withthe remainder of the device, ordinary operation of the device isinitiated by desorption of the collected sample and injection of thesample into the GC column. Alternate configurations of this aspect ofthe invention may include just a mouthpiece/straw, which acts as thesample capture device (e.g., the mouthpiece itself operates as adesorbable concentrator column).

Naturally, as technology continues to miniaturize, in due course, theportable components of this aspect and other aspects or embodiments ofthe invention or the rest of the apparatus components of this inventionwill include, e.g. a mass spectrometer on a chip, (see, for example, thehigh pressure mass spectrometer included in the M908 device availablefrom 908 Devices, Inc., 27 Drydock Ave., 7th Floor, Boston, Mass. 02210,and U.S. Pat. Nos. 8,816,272; 8,525,111; and 8,921,774) an IRspectrometer on a chip, or other versions of such technologies whichprovide enhanced portability, reduced cost, increased precision inanalysis, the ability to analyze different isotopologues included in theEBM and the like at the point of use.

6.1.8 Electronic Microcontroller

Referring now to FIG. 4D, there is provided a detailed schematic of theelectronic microcontroller subsystem 160 and the camera and displaysubsystem 170. The microcontroller 160 is in operative communication 161with the above-described disposable mouthpiece subsystem 130 (FIG. 4B),and the GC and sensor subsystem 150 (FIG. 4C), as well as the subsystemsdescribed herein below. Preferably, included in the microcontrollersubsystem 160 are the following elements: GC sensor subsystem interfaceelectronics 162, microprocessor 163, such as, but not limited to aSTM107F microprocessor, or the equivalent, now known or which hereaftercomes to be known; voltage regulators 164 for gating power from thepower subsystem (see discussion below) and transmission of appropriatelyregulated power to all other subsystems of the SMART® device; peripheraldevice interface electronics 165. Each of these elements, based oncurrent state of the art, are available as components, integratedcircuits or modules and those skilled in the art will know, based onthis disclosure, which particular components, integrated circuits ormodules are useable for the functions disclosed and described herein.The peripheral device electronics 165 controls, for example, allelements of the camera and display subsystem 170, including, but notlimited to: a WiFi, RFID, or mobile cellular data transceiver 171, orcombinations thereof, which permits communication between the SMART®device and external devices for data capture and analysis and forcommunication of control and updates to the SMART® device; aninformation display 120 associated with the SMART® device, such as butnot limited to a sixteen character, two line, backlit LCD display; avideo or still camera 172; an LED 173, such as a multicolor lightemitting diode to indicate system status and to provide a flash functionas needed when taking an image with the digital camera. Additionalperipheral devices controlled by the peripheral device interfaceelectronics 165 may include but are not limited to: memory 174, such asbut not limited to a USB memory stick or the like, EEPROM memory, orother electronic memory forms now known or hereafter developed for thispurpose; a loudspeaker 175 to provide audible alerts and/or instructionsto users of the SMART® device 100; a “Start” button 176 to activate theentire system for operation; the breath flow sensor 132; and the strawsensor 133. Each of these elements is in either two-way or one-waycommunication with the peripheral device interface electronics 165, asindicated by either two-way or one-way arrows in FIG. 4D between theseelements.

6.1.9 Power Subsystem, GPS, Wireless Communication

With reference to FIG. 4E, powering the entire device is achieved by invarious embodiments by a wall power transformer subsystem 190 alone orin operative communication with an internal rechargeable batterysubsystem 191. The wall power transformer subsystem 190 is, for example,a 90-240 volt AC, 50/60 Hz in, 9 volt DC. 1.5 amp output, preferably anIEC 60601 approved device. The internal rechargeable battery subsystem191 is, for example, composed of a pair 192 of UL approved rechargeablelithium cells (e.g., type 18650), providing 3.7 V, 2200 mAhr perbattery. In addition, included in the battery subsystem there isdesirably provided over-current protection circuitry 193,over-temperature protection circuitry, over/under voltage protectioncircuitry, and voltage regulation. Power is supplied from the wall powertransformer subsystem 190 to the internal rechargeable battery subsystem191 via an appropriate jack 194. Preferably, power supplied from thewall power transformer subsystem 190 is 9 volts DC power. Thus, whenstart button 176 is activated, the processor 160 “wakes-up” from its“sleep” state and power is provided from the voltage regulator 164 tothe peripheral device interface electronics 165 and GC subsysteminterface electronics 162 as needed for operation of the system.

The SMART® mGC can operate on rechargeable batteries, 192, which, whenfully charged, (e.g., when lithium batteries are used) providessufficient power for at least 10 complete breath measurement operationswithout the need to be recharged. In one embodiment, batteries arepermanently installed into the battery holder and are not removable,while in other embodiments, the entire battery pack is exchangeable orprimary batteries, e.g., lithium ion technology, may be used.

We envision the SMART® device will be utilized in less industrializedcountries of the world, as well as all over the globe, where wall A/Cpower supply is not always available and locating and retrieving thedevice could potentially become a problem. To overcome these obstacles,in one embodiment according to the invention, miniature recharging solarpack technology is included in the device. A GPS tracking subsystem islikewise desirably included in an embodiment of the device and isintegrated with the microcontroller 160. The internal wirelesscapability of the SMART® device allows interaction with other wirelesslyenabled devices and technologies, including, but not limited to, forexample, smart phones (iPhones, Android phones, and the like), tabletcomputers, other computers and the like.

Integrated patient/health monitoring systems and medication containersthat manage or track access to medications based on communication withthe device according to this invention are likewise optional adjuncts toor may be integrated into the system according to this invention.

6.1.10 Breath Sample and Concurrent Biometric Acquisition

Upon detecting breath flow, a microcontroller activates a small samplingpump that collects a representative breath sample for analysis(nominally 30 cc) over a pre-defined time period—preferably about afive-second time span—at a nominal flow rate of 300-400 sccm. The breathsample is collected on the thermally desorbable concentrator 201. Theexcess breath flow is vented through the flow restrictor opening on themouthpiece 136 (FIG. 5). A biometric, e.g., camera image of the subjectproviding the breath, is obtained and time stamped so that time ofbiometric and breath sample acquisition can be confirmed as beingconcurrent.

The concentrator 201 consists of a small stainless steel tube or thelike packed with a sorbent polymer (e.g, Tenax™ TA) that is commonlyused in gas chromatography to adsorb molecules of interest whileallowing molecules that are not of interest (e.g., water vapor andcarbon dioxide) to pass through the system. When the temperature of thesorbent is raised, the polymer desorbs the molecules of interest,effectively concentrating them. Once the concentrator has warmed up,valves 203, 204, 302 (FIG. 4C) are energized, causing pressurized clean,dry air from the carrier gas generator to backflush the plug of purgedmolecules from the concentrator onto the analytical column of the gaschromatograph.

6.1.11 Replaceable Ambient Air

Elements of the ambient air scrubber comprised of elements 300, 303-309and 311, (see FIG. 4C), are replaced by the manufacturer or user duringroutine maintenance or service. The carrier gas utilized in the systemis preferably generated from ambient air that is passed and cleanedthrough two different scrubbers. Of course, a portable carrier gas couldbe utilized, or the device may be linked to a conventional carrier gas,but this involves additional complexity and reduced portability whichthe present device circumvents by inclusion of the ambient air scrubberdescribed herein. The first 303 contains activated charcoal to removeorganic compounds that might be present in the ambient air and whichmight otherwise interfere with analysis of volatile organic compoundspresent in samples to be analyzed. The second 308 contains molecularsieve 13X and indicating Drierite™ to remove humidity from the air. Sodalime is useful to remove carbon dioxide. Nafion® tubing (or equivalentperfluorosulfonic acid polymer) is useful to remove water. The smallpump 304 compresses the air from the charcoal scrubber 303 and injectsit into the desiccant scrubber 308 through a small flow restrictor 307.The pressure generated by the small compressor pump 304 is monitored andcontrolled by the microcontroller 160 via the carrier pressure sensor311 to maintain a constant carrier gas flow as necessary to keep the GCcolumn 152 head pressure constant. The system operation is fullyautomatic once the breath sample has been collected. The analysisprocess takes about 180-220 seconds. When the analysis is completed, thesystem purges itself with clean air to eliminate the possibility ofbreath marker vapor carry-over and to prepare it for the next sample.

6.1.12 Data Handling

All data acquired by the SMART® GC are preferably encrypted and storedon a USB memory stick or equivalent on-board, non-volatile memory. Thispermits retrieval of data in the event of wireless communicationfailures. The on-board memory has enough capacity to store all of thedata and images associated with more than 100,000 breath measurements.

The microcontroller 160 initiates the breath sample collection processwhen the breath flow sensor 132 signal exceeds a threshold. Themouthpiece/straw sensor 133 (FIG. 4B) is, in one preferred embodiment,an optoelectronic device that emits a low intensity IR beam and detectsthe proximity of reflective objects, such as the mouthpiece. This allowsthe microcontroller to wait until the user has properly inserted thestraw 130 before advancing to the breath collection process. The breathflow sensor 132 is, in one preferred embodiment, a heated thermistorthat detects resistance changes when cooled by the flow of air passingover the sensor. Breath flow can also be sensed using a pressure sensor.

The GC detector signal 158 is digitized using a voltage-to-frequencyconverter and frequency counter in the microcontroller 160, whichprovides excellent dynamic range and noise immunity. Accordingly, alloutput signal data are reported as “counts”. A digital potentiometer,contained in the GC sensor subsystem 162, controlled by themicrocontroller 160, is used to attenuate the output voltage from theMOS detector.

During sample elution from the GC analytical column 152, the signal fromthe MOS detector 158 is logged e.g., twice each second by themicrocontroller 160. A peak-detection algorithm resident in themicrocontroller 160 locates the retention time and peak height of everycompound that elutes during the predetermined chromatographic window.When a peak is found in specific windows specified in the scriptcommands, the computer logs the successful detection of the analyte ofinterest and reports the presence of the compound that typically appearsin that window. Not only can the device detect the analyte, but it ispreferably adapted to measure absolute amounts, changes in absoluteamounts (referred to herein as the “delta” or A in the givenparameter/measurement), and to provide an assessment (e.g., a yes/noreadout) for particular compounds.

Key system status information is logged for each measurement. Thisinformation includes, but is not limited to, the elapsed run time, timesince last service, pump and oven heater duty cycles, and batteryvoltage. This allows remote assessment of the system functionality.

With reference to FIG. 14, there is provided a logic flow diagram for apreferred embodiment according to this invention. Starting from theprocessor layer 500 which has a two-way communication data flow withlayer 1, 501, comprising the various drivers for each of the device'ssub-components, including but not limited to the oven, pumps, camera,webserver, solenoids, etc. At the next level of control, there isprovided a layer 2, the SmartEngine, 502, which interprets SmartScriptcommands and invokes appropriate devices, in two-way communication withlayer 1 below 501 and layer 3 503 above. Finally, there is provided athird layer, layer 3 503, in two-way communication with layer 2 below.Layer 3 503 implements SmartScripts, permitting users and implementersof the device to program the SMART® device in plain language,implementing complex task sequences and flexibility in alteringparameters of device operation.

With reference to FIG. 15, there is provided further granularity forcomprehending the data flow and operation of one embodiment according tothis invention. According to this representation, there are threeinterconnected modules, Module A, Module B, and Module C. Module Acomprises the SMART® gas chromatograph device, including the gaschromatograph and detector which produce data which the deviceprocesses, 510, the camera and data from the camera 511, both of whichdata streams are preferably subject to encryption at 512. The data orencrypted data is then stored on an internal storage, e.g., a 1 gigabyteinternal flash storage or equivalent data storage medium, 513. Thestored data is uploaded to an embedded, preferably wireless, web server,514, for transmission to external data storage, analysis and, ifappropriate, action. This is accomplished over communication lines 515and 516, to modules B and C, where data lines 515 and 516 comprisetwo-way web (HTTPS encrypted) connections, providing data to a dataserver, 517, and end user(s) (via, e.g., a web browser or equivalentinterface), 518. Secure storage and archiving of data is accomplished inan appropriate database and secure storage system 519.

In one preferred embodiment according to this invention, there isprovided an RFID communication system whereby, on confirmation of thetaking or administration of a medication dose by a subject, a signal istransmitted from the device to a medication dispenser which is lockeduntil the next dose is due to be taken.

6.1.13 Camera and Display

The SMART® mGC incorporates a digital camera 172 and a liquid crystaldisplay 120 for visual prompts. The camera is controlled such that abiometric measurement of the subject providing the exhaled breath samplefor analysis is captured and time stamped for each collected breathsample. The camera is selected to permit accurate image capture at afocal length appropriate to the distance from the camera lens to the endof the mouthpiece where each subject interfaces with the device toprovide exhaled breath samples for analysis. In a preferred embodimentaccording to this invention, a camera is utilized which has a wide anglelens (e.g., 120 degree field of view) to ensure acquisition of areliable image even when the device is held at unusual angles by theuser.

In a preferred embodiment according to this invention, a relationship isdefined between the length “L” of the mouthpiece 130, and the focaldistance “D” of the a photographic image capture device 121 toconcurrently document the image of the subject exhaling into the device.In a preferred embodiment L=D≦5 cm. Preferably, L=D<5, 4, 3, 2 or even 1cm. This permits optimal acuity in capturing the identity of the subjectexhaling into the device without at the same time requiring use of long,cumbersome or unsightly straws/mouthpieces 130. In a preferredembodiment, a camera such as an OmniVision (Sunnyvale, Calif.), OV96551.3 megapixel camera-on-a-chip is utilized.

6.1.14 Operational Specifics of the Above Described Device and itsSubsystems

Referring now to FIG. 8, in FIG. 8A, the valving is shown for samplecollection without numbering to keep the figure clear. Reference shouldbe had to FIG. 4C for component numbering. Exhaled air enters the SMART®device via the mouthpiece 130 and is directed to the concentrator columnvia conduit 134, receiver port 151 and, via valve 203 being adsorbed toconcentrator column 201. The sample pump 300 draws the sample into theconcentrator 201 and vents air stripped of molecules of interest. Theadsorption is conducted at a reduced temperature, such as 25 degreescentigrade. In FIG. 8B, the concentrator 201 is heated to an elevatedtemperature, such as 150 degrees centigrade, to thermally desorb thebreath borne molecules that have been trapped on the concentrator 201.In order to direct desorbed molecules from the concentrator through theGC column 152, valve 203 at the proximal end of the concentrator 201 isclosed to the mouthpiece, but opened to the GC column 152. Ambient airis drawn through the scrubber 303 by the carrier pump 304, through theflow restrictor 307 and through the second scrubber 308 and valve 302for delivery to the distal end of the concentrator column 201 via valve204, thereby driving the desorbed molecules from the concentrator 201into the mGC column 152 through the detector 158 and, finally, out thevent 159. See FIG. 9 and Example 2 for a typical chromatogram producedby this system.

In FIG. 10A, there is shown a photographic representation of theinternal components and architecture of a first exemplary embodiment ofthe SMART® device according to this invention. Visible in thisphotograph are at least the following components: battery pack 192;external power connector 194; battery pack connector 192 b; USB solidstate memory 174; replaceable dessicant-sieve cartridge 308; sample pumpfor breath sample collection 304; scrubber air pump vent 304 b; flowrestrictor 307; carrier gas pump which pressurizes the scrubber 300;charcoal scrubber 303; and the scrubber air inlet port 303 b. In FIG.10B, there is shown a sling 320 for holding, in one preferred embodimentaccording to this invention, the dessicant-sieve cartridge 308 andcharcoal scrubber 303 in flexible but firm position. The sling 320comprises a preferably elastomeric material comprising perforationstherein 321 and 322 through which the dessicant-sieve cartridge 308 andcharcoal scrubber 303

In FIG. 11, the obverse view from that shown in FIG. 10 is provided as aphotographic representation of the internal components and architectureof a first exemplary embodiment of the SMART® device according to thisinvention. Visible in this photograph are at least the followingcomponents: attachment; breath inlet port 134 c; concentrator column201; line to sample pump 300 b; scrubber air lines 309 b; GC column oven153; fan 205; vent 159 from GC detector 158.

In FIG. 12, there is shown the air filter path in an exemplaryembodiment according to the invention. Shown in this figure are: thescrubber air pump 304 which draws ambient air in through port 303 b intothe charcoal scrubber 303, via carrier pump coupling 305, past thescrubber pump pressure sensor port 311 b, through flow restrictor 307,then through the desiccant scrubber 308 and from there out port 309 intothe valving leading to the GC column.

Naturally, those skilled in the art will appreciate that these variouselements are shown as an exemplary layout in one embodiment according tothe invention and different or equivalent layouts and componentdimensions are conceivable by those skilled in the art based on thedisclosure provided here. In addition, not all components described inthe general description are labeled in this figure—such as the PCB onwhich all the above components are laid and interconnected and theactual GC column which is obscured in these photographs by othercomponents.

Those skilled in the art will further appreciate from the presentdisclosure that each of the elements shown herein may be furtheroptimized by further miniaturization, such as, for example, through theuse of micro-pneumatics.

6.1.15 Smart® Device User Interface and Sequence of Operation

Those skilled in the art will appreciate, based on the presentdisclosure, that there are wide array of variations to this component ofthe invention which may be utilized without departing from the core ofthis invention. Thus, for example, in one embodiment, a single breathcollection is all that is required, because essentially no backgroundexists. In other embodiments, an initial breath is obtained prior tomedication being taken or administered followed by a second breaththereafter, for each dose of medication.

a. Baseline Breath Sample Acquisition:

It will be appreciated, based on the full disclosure provided herein,that a baseline breath sample may not be required in certain embodimentsof this device used in connection with particular combinations of AEMsin various AMAM, IMAM or CMAM applications. Thus, for example, wherevery low background of a particular EBM is known to occur, a baselinebreath may be dispensed with. As a specific example of such a scenario,consider the embodiments of this invention relating to use of i-AEMswhere essentially no background exists for particular i-EBMs. Where abaseline breath is considered of value, this is obtained as describedhere.

With reference to FIG. 13, in one embodiment according to thisinvention, at 400, the device is woken by pressing the start button 176,which initiates a startup routine at 401, a battery display to show theuser whether the device has sufficient power to operate properly, 402,and if so, the device displays a message that it is initializing 403.The device then initializes all settings to a starting condition readyfor exhaled breath sample receipt 404. “ATTN” 404 a in the figure refersto an audible signal to alert the user that action is required. The useris then prompted 405 to insert a new, clean mouthpiece “straw”. Amouthpiece insertion subroutine is then initiated 406 which, if nomouthpiece is detected, prompts the user to insert the mouthpiece 405,or, the system times out 406 a after a pre-set time, optionally about 1hour, if no mouthpiece is inserted within the preset time period. Oncecorrect mouthpiece insertion has occurred, this is confirmed to the user407, and the user is advised 407 a that, prior to taking a medication orstudy capsule, to blow/exhale into the mouthpiece, 408. A breathdetection subroutine 409 initiates to confirm detection of breath beingexhaled into the device (triggered by the flow sensor 132). If no breathis detected, the system times out after a short while, optionally about4 minutes. If a breath is detected, a biometric measurement of the useris captured, such as a fingerprint, or, preferably, a photograph istaken, 410 a, and the user is prompted 410 b to continue to blow intothe mouthpiece until the device detects that a sufficient amount ofbreath has been detected 411. When a sufficient amount of breath hasbeen detected, the subject is prompted with a “good job” or similarprompt 412 to indicate that a sufficient breath sample has beencollected for analysis.

b. Sample Breath Acquisition to Confirm Medication Adherence

Once the device has confirmed that a sufficient pre-medication baselinesample has been acquired 411, the subject is then prompted to take theirmedication, study capsule or whatever dosage form it is for whichmedication adherence monitoring is being conducted 413 a and the user isprompted 414 to press the start button 176 when the medication has beentaken. The device then enters a subroutine 415 to confirm that the userhas pressed the button. If no button press is detected within a presettime, e.g., thirty minutes to an hour, the device times out 429 and goesto sleep 430. However, if the button press is detected at 415, theroutine continues, by a prompt 415 a advising the user to please wait apre-set amount of time, (a time optimized for the vast majority ofsubjects in clinical testing, depending on the medication/AEMcombination in use, typically from about five minutes to about an hour,and preferably about ten to about twenty minutes). To ensure that thesubject waits the optimal amount of time after taking the medication andto prevent the subject from forgetting to provide a post-medicationbreath, a countdown timer routine 415 b initiates. During that period,the device warms up in readiness for receipt of the breath sample postmedication 416 during which time the subject continues to wait for thefull optimal time period for post medication breath collection 417.“ATTN” in the FIG. 418 refers to an audible signal to alert the userthat action is required. Following this, the user is again prompted toblow into the mouthpiece 419. A breath detection subroutine initiates420. If no post-medication breath sample is detected, the device is setto time out 420 a within a pre-set time period, say about 1 hour.However, if a breath is detected, as before, a biometric is captured,preferably a photograph 420 b, and the user is prompted to keep blowing421 until a sufficient breath sample 422 is detected. When a sufficientamount of breath has been detected, the subject is prompted with a “goodjob” or similar prompt to indicate that sufficient breath has beencollected 423. At this stage, the sample collection procedure has beencompleted and the user is prompted to remove the used mouthpiece 424. Abrief period, e.g., five seconds, is provided 425 for the device toconfirm that all operations have been successfully completed, at whichpoint, the user is prompted to advise that the breath samples have beenproperly collected, by display of a message, e.g., “HAVE A GREAT DAY!”or the like, 426. The second breath sample is analyzed by the device427, with calculation of changes (delta, A) in key analytes (e.g.,2-butanone), and the results are uploaded 428 to a database, locallyand/or at a remote site, where medication adherence is optionallychecked, confirmed or otherwise evaluated, either automatically or by anappropriate responsible party. At this stage, the device preferably goesinto a sleep state, 430.

In light of the forgoing disclosure, those skilled in the art willappreciate that the present invention provides a novel SMART® device forSelf Monitoring and Reporting Therapeutics. The Type I Self Monitoringand Reporting Therapeutics (SMART®) device comprises a miniature,portable, gas chromatograph subsystem for separation and analysis ofcomponents of a breath sample provided by a subject. The gaschromatograph is, preferably, provided in the device in combination witha separated compound sensor appropriate to detection and/or quantitationof the particular exhaled breath component of interest (VOC, EBM), andat least one or a combination of any one or a combination of:

a. means for subject biometric measurement for definitive identificationof a subject concurrent with the subject providing an exhaled breathsample;

b. a breath flow sensor;

c. a wireless data transceiver;

d. a mouthpiece either with ports for breath sampling, venting, correctemplacement confirmation, and excess breath venting, or, a simple tubewith a mouthpiece receiver port bearing features as described above foraccepting the mouthpiece, breath sampling, venting, and emplacementconfirmation;

e. a breath detection and sampling subsystem in operative coupling withthe mouthpiece;

f. a disposable air scrubber;

g. a rechargeable battery pack subsystem; and

h. a microcontroller subsystem in operative electrical coupling with atleast one, several, and preferably all electrical components of elements(a)-(f).

It will further be appreciated that the SMARM device according to thisinvention may comprise, in various embodiments, any one or combinationof the following:

a. the mouthpiece comprises: an exhaled air inlet; a breath flow sensorport; a breath sample conduit receiver port; and a vent; or simply aninlet and an outlet;

b. the breath detection and sampling subsystem in operative couplingwith the mouthpiece comprises: a mouthpiece receiver comprising: abreath sample conduit for operative coupling with the breath sampleconduit receiver port; a breath flow sensor for operative coupling withthe breath flow sensor port; and a mouthpiece sensor for detection ofproper insertion of the mouthpiece into the mouthpiece receiver;

c. the disposable air scrubber comprises an activated charcoal filter, adessicant, or both;

d. the gas chromatograph is included in a subsystem in operativecoupling with the breath detection and sampling subsystem and thedisposable air scrubber comprises a thermally desorbable concentratorcomprising: a thermally desorbable concentrator column; a proximal and adistal three-way valve on either end of the desorbable concentratorcolumn; a heating element in intimate association with the thermallydesorbable concentrator column; and a gas chromatograph column with adetector at the distal end thereof;

e. the wireless data transceiver subsystem comprises a WiFi, mobilecellular data transceiver, or both;

f. the biometric measurement means comprises a camera and displaysubsystem comprising a still or video digital camera which records animage of the subject at the time that the subject exhales into themouthpiece;

g. the rechargeable battery pack subsystem comprises lithium batteries;and

h. the microcontroller subsystem in operative electrical coupling withat least one and preferably all electrical components of elements(a)-(f) comprises a microprocessor, a voltage regulator, peripheraldevice interface electronics and GC sensor interface electronics.

Exemplary support for use of the Type I device as described herein isprovided in the Examples below, (in particular, but not exclusively, inExamples 1-4). It will further be appreciated by those skilled in theart that the SMART® device may include one, different combinations of,or every element (a)-(h) as listed above. In addition, the Type Iembodiment of the SMART® device according to this invention may includeand incorporate components and elements of other SMART® deviceembodiments as described herein below.

The Type I embodiment of the device, according to the invention, isexcellent for measurement in the exhaled breath of AEMs (which mayappear in the exhaled breath) and/or EDIMs (or EDEMs, that is theExhaled Breath Marker, EBM, which is typically a modified form of or ametabolite of the AEM) which appear shortly after ingestion of orapplication onto a subject of an AEM. This embodiment of the device isprimarily adapted for AMAM, but, depending on the longevity of the EDIMin the exhaled breath, the signal to noise ratio and the total mass ofAEM utilized, this device may also provide IMAM and CMAM options. Thisis described further in section 7, relative to specific AEMs andcompositions of matter for delivery of AEMs, and section 8, relating tothe SMART® system, in which particular AEM and device embodiments arematched to achieve particular AMAM, IMAM, and CMAM objectives. Exemplarysupport is also provided herein below in section 9 of this patentdisclosure.

6.2 Detailed Description of a Second Embodiment (Type II) of theImproved Smart® Device:

For the most part, the description of type I of the device in section6.1 above, is directly applicable without change or minimal change to adescription of a Type II device, unless differences/changes to aparticular element or subsystem is specifically described herein belowin this section. The key modifications in a Type II device as comparedto a Type I device are described in detail in this section. The ways inwhich such modifications enable use of different AEM compositions ofmatter, methods of medication adherence, extension of the time formedication adherence monitoring in acute medication adherence monitoringand into intermediate and chronic adherence time frames and options forIMAM and CMAM, as well as alternate SMART® systems, are described hereand in sections 7, 8 and 9.

The present invention is directed to the provision of new technology forassessing and improving medication adherence, which remains a criticallyimportant health care priority in multiple clinical settings, includingpharmaceutical drug trials, management of major diseases (e.g.,schizophrenia, diabetes, hypertension), and in the fight againstdiseases that threaten global health (e.g., TB, HIV/AIDS). In terms ofaccurately documenting adherence, methodologies, including electronicones (e.g., pill counters, electronic medication caps and the like),developed to solve this problem, have been inadequate to date, sincenone detect or document actual drugingestion/administration/application. Outside of clinical trials andresearch studies investigating strategies to improve adherence, it isunfortunately measured all too frequently, especially in the clinicalsetting, by simply questioning patients on their use of medication overthe prior several (e.g., 4 or 7) days, or prior weeks or month. Toaddress these shortcomings in assessing adherence, the present inventorsprovide methods, compositions and devices for achieving breath-basedMedication Adherence Monitoring System (MAMS).

The goal of Xhale, Inc's., medication adherence monitoring program is toemploy unique chemistry-based technologies to document adherence to oraland other medications. The adherence system, in a preferred embodiment,comprises a smart-phone sized sensor device or smart-phoneinter-operable accessory, using breath as the diagnostics matrix tomeasure metabolites of generally recognized as safe (GRAS) foodcomponent-based taggants, that is, the Exhaled Breath Marker (EBM,whether that be an EDIM or EDEM). In other words, GRAS foodadditive-based taggants are used to document when a dosage form (e.g., apill) entered the gastrointestinal tract or entered anotherphysiological compartment (e.g., via transdermal, intranasal, vaginal,rectal, or other mode of delivery), was absorbed into the blood, and wasmetabolized to taggant metabolite(s) detectable in the exhaled breath, aprocedure called definitive medication adherence. In contrast, we havealso investigated presumptive medication adherence, where we candocument that a medication was placed in the mouth of a subject but notactually swallowed. However, certain patients (e.g., schizophrenics,court ordered TB patients on drug therapy, so-called “professional”patients or deceptive patients enrolled in clinical trials) can bedeceptive about whether they actually swallowed their medications. TheMAMS methodology to which the present invention applies is of thedefinitive type.

Through human testing, we have identified a number of GRAS foodadditives, referred to herein as taggants (AEMs) that appear to have theappropriate features (e.g., generation of EDIMs) for an effectivebreath-based MAMS of the definitive type. One major strategy to achievethis goal is to utilize isotopically-labeled chemicals, preferably GRAScompounds, and particularly deuterated ones, which generate deuteratedEDIM(s) (i.e. i-EBMs/i-EDIMs) in breath to document definitivemedication adherence.

6.2.1 Key Differences Between Type II and Type I Devices According tothis Invention

According to this embodiment of the invention, the Type I devicedescribed in section 6.1 above is modified to enable use of AEMscomprising non-ordinary (but preferably stable, i.e. non-radioactive)isotopes in the AEM. Such AEMs are referred to herein as i-AEMs, and aremanufactured and selected for use with this embodiment of the devicesuch that EDIMs which are produced following ingestion or application ofthe i-AEMs include the non-ordinary isotope, and are, therefore i-EDIMs.Accordingly, this section of this patent disclosure describes andenables methods of making and using medications, medicinal compositions,devices, and systems and for production and detection of, in exhaledbreath of a subject, volatile organic compounds (VOCs) which includenon-ordinary, but preferably stable (non-radioactive), atomic isotopes,referred to as i-EBMs, (Exhaled Breath Markers containing at least onenon-ordinary, but stable (non-radioactive) isotope), for definitivemedication adherence monitoring. A key difference between a Type Idevice and a Type II device according to this invention is that, whilefor a Type I device, a simple sensor such as a MOS sensor suffices, forthe Type II device, an infrared (IR) sensor is preferred. Another keydifference is that in one embodiment of the Type II device, preferably,the device includes a catalytic combustion chamber to convert VOCs intowater and carbon dioxide. Inclusion of the catalytic incineratorsimplifies detection in this embodiment of the device by allowing the IRdetector to be tuned to the particular non-ordinary isotope sought to bedetected, whereas, without the catalytic incineration component, atunable IR sensor may be required to permit the device to be tuned todetect different VOCs of interest. Thus, inclusion of the catalyticincinerator essentially converts a particular IR sensor into a universalIR sensor. For clarity, in the present invention, the catalytic elementfor IR applications is only required where an IR detector is sought tobe used in the same manner as described above for use of a MOS detectorin an mGC-MOS embodiment of the device. Further details and descriptionon these aspects of the invention are found herein below.

In this embodiment according to this aspect of the invention, there isprovided a device and a method of using the device, for detecting in anexhaled breath sample a VOC comprising a non-ordinary but stable(non-radioactive) atom, e.g., deuterium, wherein, in a preferredembodiment, the device comprises:

-   -   (a) means for separating VOCs in the exhaled breath;    -   (b) means for stripping the exhaled breath sample of moisture,        (e.g., using Nafion® tubing or similar perfluorosulfonic acid        polymer), carbon dioxide, (e.g., using soda lime) or both;    -   (c) means for converting VOCs in exhaled breath into carbon        dioxide, water, or both, such that said non-ordinary but stable        (non-radioactive) atom (e.g., deuterium or other non-ordinary        isotope) included in the VOC is included in the water fraction,        carbon dioxide fraction, or both, such that, e.g deuterated        water, isotopically labelled carbon dioxide (with stable carbon        or oxygen isotopes) is produced and available for quantitation        in the exhaled breath sample.

6.2.2 Additional Definitions and Abbreviations Relevant to this Aspectof the Invention:

SMART®—Self Monitoring and Reporting Therapeutics—embodiments of adevice, medication, composition, system and method wherein adherence bya subject in taking/receiving a medication is facilitated by detecting avolatile molecule in the exhaled breath of a subject, wherein thevolatile molecule only appears in the exhaled breath of a subject aknown period of time and at a known concentration after a medication istaken by the correct person, at the correct time, at the correct dose.

i-SMART®, as for SMART®, but including compositions, methods, systemsand devices optimized for detection of compounds in the exhaled breathwhich include a non-ordinary, but preferably stable (non-radioactive)isotope, as further described herein below; those skilled in the artwill appreciate that it is the presence of an isotope at an abundancethat is completely different than the abundance of the isotope as itoccurs in nature (due to that isotope having been selected for inclusionin the i-AEM) that is detected in this mode of practicing thisinvention, and any detector or sensor now known in the art or whichlater comes to be know which is sufficiently sensitive to detect theparticular isotope of interest and its abundance (concentration) may beused for this purpose.

API—Active Pharmaceutical Ingredient—the medication or medications withthe therapeutic effect of which is desired to be delivered to subjectsand the administration of which is to be monitored via the SMART® ori-SMART system.

i-API—An API containing at least one non-ordinary, but stable(non-radioactive) isotope of hydrogen (i.e. deuterium), carbon (e.g.,C¹³), or the like, and which, on introduction into a living subject,results in the production of at least one i-EBM. This is typically as aresult of the metabolism of the i-API to produce a cognate i-EBMspecific to that particular i-API. In some cases, the i-API itself maybe the i-EBM—e.g., deuterated propofol would appear in the exhaledbreath, as does non-deuterated propofol. It will be appreciated that notthe entire fraction of the API need contain the non-ordinary isotope,and that fraction that does is referred to herein as the i-API fraction.

EBMs—Exhaled Breath Markers—molecules which appear in the exhaled breathfollowing ingestion or other form of administration of a medicationcontaining a marker which gives rise to the EBM.

i-EBMs—Exhaled Breath Markers (including EDIMs and EDEMs) containing atleast one non-ordinary, but stable (non-radioactive) isotope of hydrogen(i.e. deuterium), carbon (e.g., C¹³), or the like.

EDIM—Exhaled Drug Ingestion Marker—a molecule detected in the exhaledbreath of a subject who has ingested a medication (drug) which includes,as part of the Active Pharmaceutical Ingredient (API) or as part of aseparate molecule packaged for co-delivery with the API.

i-EDIM—an EDIM comprising at least one non-ordinary but non-radioactive(i.e. stable) isotope.

EDEM—Exhaled Drug Emplacement Marker—a molecule detected in the exhaledbreath of a subject who receives a medication (drug) which includes, aspart of the Active Pharmaceutical Ingredient (API) or as a separatemolecule packaged for co-delivery with the API, when received by a routeother than ingestion.

i-EDEM—an EDEM comprising at least one non-ordinary but non-radioactive(i.e. stable) isotope.

AEM—Adherence Enabling Marker—a molecule included in a medication whichaccording to this invention gives rise to EBMs in the exhaled breath ofsubjects who have taken or been administered the medication includingthe AEM.

i-AEM—An AEM which includes at least one non-ordinary butnon-radioactive (i.e. stable) isotope which according to this inventiongives rise to i-EBMs in the exhaled breath of subjects who have taken orbeen administered the medication including the i-AEM.

ADME—Absorption, Distribution, Metabolism, Excretion.

Ordinary and non-ordinary isotopes non-radioactive isotopologue of agiven element that is the dominant form in nature; the dominantnon-radioactive isotopologues, termed the “ordinary isotopologues” arebolded, while the non-ordinary isotopes are not bolded: Hydrogen atom:¹H (protium), ²H or D (deuterium); Carbon atom: ¹²C, ¹³C; Nitrogen atom:¹⁴N, ¹⁵N; Oxygen atom: ¹⁶O, ¹⁷O, ¹⁸O; Sulfur atom: ³²S, ³³S, ³⁴S, ³⁶S.Sensors are known in the art for measuring and distinguishing theseisotopologues with exquisite sensitivity (at levels as low as low partsper trillion).

Where a particular combination or permutation of elements is describedin connection with a particular embodiment or element of the presentinvention, those skilled in the art will appreciate that suchcombination or permutation of elements may be applicable to any otherembodiment of the present invention, unless specifically excluded or,from the given context, this is clearly not appropriate. Thus, forexample, in describing herein below formulations, dosage forms,compositions and methods for topical, vaginal or rectal delivery ofAPIs, i-APIs, and i-AEMs, considerations relevant to quantities of i-AEMdelivery, type of i-AEM delivered, separation of the i-AEM from the API,etc. for such mode of delivery are applicable to any other mode ofdelivery; however, from the context of the description, it is clear thatdifferent formulations would be appropriate for each mode of delivery.

6.2.3 Rationale for Use of Isotopic Labeling to Confirm MedicationAdherence

The isotopic labeling of molecular entities that serve as substratesthat, via enzymatic degradation or other processes, liberateisotopically-labeled i-EBMs, is a critically important strategy towarddesigning and developing an optimal MAMS—particularly for IMAM and CMAMembodiments. In a series of experiments (FIGS. 23-42) using a gas phaseFourier Transform Infrared (FTIR) device (Nicolet 6700 FT-IR, 5 literBreath Sample, 22 meter path length) using human breath and a nitrogenenvironment, we confirmed key scientific assumptions, which underlie theadvantages listed above for isotopic labeling in MAMS. Specifically, weinvestigated the effect of non-ordinary isotopic (e.g., deuterium, ¹³C;see Table 1) labeling on the FTIR spectrum of key alcohols, aldehydesand ketones, relative to those containing ordinary isotopes at roomtemperature. Important findings include:

1) FTIR poorly discriminates between deuterated and ordinary alcohols ofsimilar structure; the FTIR absorption spectra for ordinary methanol andethanol as well as deuterated methanol and ethanol are very similar.

2) FTIR spectra for a given alcohol (ordinary vs deuterated) is highlydistinctive and can be used to discriminate among them (i.e., CD₃-OH vsCH₃—OH or CD₃D₂-OH vs CH₃CH₂—OH) (see FIG. 66). In contrast, GC-MS caneasily distinguish between all these species. The miniature gaschromatograph (mGC) can easily distinguish between specific alcohols ofdifferent carbon number but not among deuterated and non-deuteratedalcohols with the same number of carbons.

3) FTIR does not provide a high degree of discrimination betweendeuterated and ordinary aldehydes of similar structure; the FTIRabsorption spectra for ordinary formaldehyde and acetaldehyde as well asdeuterated formaldehyde and acetaldehyde are similar. 4) FTIR spectrafor a given aldehyde (ordinary vs deuterated) is highly distinctive andcan be used to discriminate among them (e.g., CD₃CDO vs CH₃CHO or CD₂Ovs CH₂O). Taken together, the results indicate that isotopic labelingshows great promise in the specific and sensitive detection of i-EBMs inhuman breath for MAMS.

From the FTIR experiments, it appears that three fundamentally differentdeuterated i-EBMs could be distinguished by utilizing a tunable midIRlaser with a center wavenumber of 2150±10% variability (wavenumberrange: 2000 to 2300 cm⁻¹). These EBMs include: 1) carbonyl (i.e.,acetone with per-deuterations on methyl groups)—wave number=2040 cm⁻¹;2) aliphatic (i.e., hexane with per-deuterations on terminal methylgroups—wavenumber 2240 cm⁻¹; and 3) aromatic (e.g., benzaldehyde,cyclopentanone, or cyclohexanone with per-deuterations on thering—wavenumber 2290 cm⁻¹.

Deuterium, depending upon the class of molecules they are placed on, thenumber of deuterations on a molecule, and their proximity to variousbond types (e.g., amine, sulfhydryl, aromatic, etc.) on the molecule,can provide various types of molecular entities with unique analytical“signatures” in various biological media, including but not limited tobreath, blood, urine, sweat or saliva. Various analytical techniquessuch as IR or mass spectroscopy can be used to not only distinguishdeuterated parent compounds from their deuterated metabolites (both inthe gas and/or liquid states), but can also easily discriminatedeuterated molecules from those identical natural compounds containingordinary hydrogen (e.g., ethanol versus deuterated alcohol; aldehydeversus deuterated aldehyde; methanol versus deuterated methanol). Itsuse will reduce the need or even eliminate the step of obtainingbaseline breath samples, as well as markedly simplify the FDA regulatoryprocess for new drugs allowing for faster time to market withinexpensive and reliable technology. Deuterated compounds are generallyregarded as nontoxic and as having the same (or very similar)pharmacodynamic and pharmacokinetic properties as their undeuteratedparent compounds. Last, deuterated approaches can be used to potentiallymonitor the metabolism of many important therapeutic agents.

From these studies, we conclude that the use of primary alcohols astaggants for oral adherence is not ideal. They generate aldehydes, whichare very rapidly converted to their corresponding carboxylic acid. It isdifficult to measure primary alcohols or their metabolites in the breathof humans following oral ingestion. The use of secondary alcohols astaggants for oral adherence appears very promising. They generateketones, which persist in the breath of humans, following the oraladministration of secondary alcohols. We are currently focusing onseveral secondary alcohols, including but not limited to 2-butanol,isopropyl alcohol (IPA, isopropanol), and 2-pentanol. These generate2-butanone, acetone and 2-pentanone, respectively, which, other thanacetone, are present in very low concentrations in the baseline breathof humans. Isopropyl alcohol is considered to be an excellent taggant,which will generate the ketone, acetone. In addition to incorporatingsmall quantities of isopropyl alcohol into capsules or tablets, a greatvariety of GRAS isopropyl-based esters, which would generate isopropylalcohol via esterases, exist in the food database. From the FTIRexperiments, it appears that three fundamentally different deuteratedi-EBMs could be distinguished by utilizing a tunable midIR laser (tomeasure C-D vibrational stretch) with a center wavenumber of 2150±10%range (wavenumber range: 2000 to 2300 cm⁻¹). These EBMs include: 1)carbonyl (e.g., acetone with per deuterations on methyl groups)—wavenumber=2040 cm⁻¹; 2) aliphatic (e.g., 2-butanone with deuterations onnon-alpha carbons—wavenumber 2240 cm⁻¹), and 3) aromatic (e.g.,benzaldehyde, with per deuterations on ring—wavenumber 2290 cm⁻¹. Bycombining molecules with molecular attributes including carbonyl,aliphatic and/or aromatic properties, up to 6 different types ofmolecules could be readily detected using tunable or non-tunable mIRapproaches: 1) carbonyl only, 2) aliphatic only, 3) aromatic only, 4)carbonyl+aliphatic, 5) carbonyl+aromatic; 6) aliphatic+aromatic, and 7)carbonyl+aliphatic+aromatic. With the use of other types of opticaldetection systems, including but not limited to quantum cascade lasers,lead salt lasers, frequency-combed based systems, cavity-enhancedoptical frequency comb spectroscopy, mode-locked femtosecond fiberlasers, and virtually imaged phase array (VIPA) detectors, a very largenumbers of analytes, particularly in the breath, could be potentiallydetected.

Metabolic considerations are shown in greater detail in FIGS. 22 and43-53 to assist in describing, and enabling those skilled in the art, inthe practice of designing appropriate i-AEMs to achieve inclusion ofnon-ordinary isotopes in the i-EBMs. FIGS. 23-42 are provided to showthe power of FTIR to distinguish signals obtained from ordinary andnon-ordinary isotopes in different candidate i-EBMs, depending on thenature and degree of non-ordinary isotope substitution in select i-AEMS.Further details regarding these figures are provided in the Examplessection included herein.

6.2.4 Type II SMART® Device for MAM Using i-EBMs

In light of the foregoing, it will be appreciated by those skilled inthe art that, according to this invention, known methods, devices,systems and compositions for medication adherence monitoring, medicationtracking and counterfeit medication detection are improved by:

A. provision of medications comprising a SMARM medication comprising anActive Pharmaceutical Ingredient (API or i-API) alone or in combinationwith at least one non-toxic, preferably Generally Recognized as Safe(GRAS) volatile organic compound (VOC) or incipiently volatile organiccompound, the i-EBM (including i-EDIMs and i-EDEMs), preferably a directfood additive, wherein at least one atom of said i-AEM is replaced witha non-ordinary, stable (non-radioactive) isotope, such that, onadministration (ingestion, topical application, or other means ofdelivery) of the medication or a component thereof comprising thelabeled VOC, or a metabolite thereof comprising the non-ordinaryisotope, the i-EBM, is entrained and is detectable in the exhaled breathor other bodily fluid;

B. provision of a device for detecting in an exhaled breath sample a VOCcomprising a non-ordinary isotope, (the i-EBM) wherein, in a preferredembodiment, see further discussion below, the device comprises a meansfor stripping the exhaled breath sample of moisture, a catalyst forconverting the VOC to carbon dioxide and water, such that thenon-ordinary isotope from the VOC is included in the water or CO₂fraction, such that, following catalysis, e.g., deuterated water or CO₂containing isotopes of carbon or oxygen are detected and quantitated inthe exhaled breath sample; and

C. provision of a method for medication adherence monitoring whichcomprises providing a SMART® medication as described above to a subjectand using the device as described above to detect and quantitate i-EBMswithin the exhaled breath of the subject.

In a preferred embodiment according to this invention, a VOC, preferablyselected from, but not limited to, the group consisting of secondary andtertiary alcohols in which for example hydrogens are replaced withdeuterium atoms, or oxygen or carbon atoms are replaced by stablenon-ordinary isotopes, is included in a medication for ingestion ordelivery by other means (transdermal, vaginal, rectal, etc). The presentinvention demonstrates that, while kinetics of appearance of e.g.,deuterated VOCs in the exhaled breath differs depending on the route ofadministration, whether delivered orally, transdermally, or via anotherroute of delivery, and depending on the precise nature of the moleculein which deuterium is included, deuterated VOCs are readily detectablein the exhaled breath and are, therefore, excellent markers todefinitively confirm medication adherence, to track medications use andto detect and preferably prevent medication diversion or counterfeiting.

In an embodiment of the device according to this invention for detectingi-EBMs produced from e.g., deuterated AEMs or AEMs containing othernon-ordinary but stable isotopes, (i.e. i-AEMs) or metabolites thereofin the exhaled breath, there is provided a miniature portable gaschromatograph, similar to but an improvement over a first generationminiature GC device described in Morey et al., “Measurement of Ethanolin Gaseous Breath Using a Miniature Gas Chromatograph”, J. Anal.Toxicol., Vol. 35, p. 134-142, (2011). The improvements in the presentdevice include, but are not limited to, inclusion of a forward facingcamera which is synchronized with breath sample acquisition to ensurethat the breath sample and the identity of the subject providing thebreath sample (e.g., by photographic identification) are concurrentlytime-stamped; and adaptation for maximum efficiency in detectingnon-ordinary isotopes.

In a preferred embodiment of the device according to this aspect of theinvention, included in the device are the following additional elements:

a. a sample de-humidification means and CO₂ stripper through whichexhaled breath samples are passed to remove all water (including anybackground deuterated water which might interfere with subsequentquantitation of deuterated water following catalysis of VOCs to waterand carbon dioxide); b. a catalyst for conversion of VOCs in the exhaledbreath sample to H₂O or D₂O and Carbon dioxide (see, for example, EltronResearch & Development Inc., and their U.S. Pat. No. 6,458,741 Catalystsfor Low-Temperature Destruction of Volatile Organic Compounds in Air;U.S. Pat. No. 6,787,118 Selective Removal of Carbon Monoxide; U.S. Pat.No. 7,329,359 Application of Catalysts for Destruction of OrganicCompounds in Liquid Media; U.S. Ser. No. 12/257,811 A Metal Oxide Systemfor Adsorbent Applications; (seehttp://www.eltronresearch.com/docs/Low_Temp_VOC_Catalyst.pdf); see,also, “Development of Low Temperature ActiveCatalysts for CO and VOCAbatement”, Monika Molin, Department of Chemical Engineering, LundUniversity, Sweden (Jun. 27 2011, available athttp://www.chemeng.lth.se/exjobb/E605.pdf); see, further, “VOC oxidationover MnOx-CeO₂ catalysts prepared by a combustion method”, DimitriosDelimaris and Theophilos Ioannides, Applied Catalysis B: Environmental,Volume 84, Issues 1-2, 25 Oct. 2008, Pages 303-312; see, also, “Lowtemperature oxidation of volatile organic compounds using gold-basedcatalysts”, Kwenda, E., http://hdl.handle.net/10539/10408;

b. a non-ordinary isotope detector, preferably a D₂O detector.

6.2.5 Detailed Description of the Type II Device According to thisAspect of the Invention

Referring now to FIG. 19, there is shown a schematic wherein anembodiment 1000 of the device according to this invention is shown. Abreath sample, 1001, comprising 1002 CO₂, H₂O in the form of watervapor, volatile organic compounds (VOCs), and the included ExhaledBreath Marker (i-EBMs) comprising at least one non-ordinary isotope, isintroduced into the Stage 1 of the device, 1010. This stage of thedevice is for sample collection and analyte isolation, from which onlyVOCs in the exhaled breath and the i-EBMs comprising the non-ordinaryisotope, is released into Stage 2 of the device, 1020, where analytedetection and data collection occurs. According to this figure, furtherdetail is provided with respect to Stage 1, 1010, where, after thebreath sample 1001 (comprising CO₂, H₂O in the form of water vapor,volatile organic compounds, and the included exhaled breath marker(i-EBMs) comprising at least one non-ordinary isotope, 1002), isintroduced into the device, the introduced breath sample receives any ofseveral different treatments, e.g., A, B, C, or modifications,variations, permutations., equivalents and/or combinations thereof.

Thus, referring again to FIG. 19, in treatment A of Stage 1 (1010), thebreath sample 1001 (comprising water, carbon dioxide, VOCs and thei-EBM(s)) is passed through a dryer/scrubber 1011, which removes all orsubstantially all of the water and carbon dioxide endogenous to theexhaled breath sample 1001. An alternative or additional approach isshown in treatment B of Stage 1 (1010), wherein the breath sample 1001is directed into a concentrator 1012 (e.g., a tenax column or the like)which binds VOCs including the i-EBMs, but not water or carbon dioxide,which merely flow through the concentrator and are vented to theatmosphere, while retained i-EBMs are, for example, thermally desorbedfrom the column after these contaminants have been removed. In treatmentC of Stage 1 (1010), the breath sample 1001 is introduced into aconcentrator 1012, as in treatment B, but, in addition, the retainedmaterials are fractionated via a fractionation means, e.g., achromatographic column. In a preferred embodiment, the chromatographiccolumn is a miniature gas chromatography (GC) column, thus making itpossible for the entire device to be portable. The concentrator, 1012,is preferably a material which efficiently retains VOCs, includingi-EBMs, while allowing all other breath components to flow through(i.e./e.g., moisture, carbon dioxide), but is easily desorbed ofretained VOCs/i-EBMs, e.g., by application of heat. Of course, both adryer/scrubber 1011 and concentrator 1012 may be utilized in series toensure removal of all water and carbon dioxide endogenous to the exhaledbreath sample, prior to further treatment (GC column separation andStage 2 treatment).

Also shown in the figure is further detail of the Stage 2 of the device,1020, whereby the i-EBMs emergent from Stage 1 can be treated by atreatment such as treatment A or Treatment B in Stage 2 or inequivalents, modifications, permutations or combinations thereof. InStage 2, treatment A, the i-EBMs 1003 are directly passed through aninfra-red (IR) detector and the signals obtained from passage of thesample through the detector is collected and analyzed. In Stage 2,treatment B, the i-EBMs are subjected to catalytic combustion 1022, toproduce carbon dioxide and water from the i-EBMs and VOCs. Wherenon-ordinary isotopes are included in the i-EBMS, these appear in thecarbon dioxide or water (deuterium oxide) fraction and are then passedthrough an IR detector for data collection and analysis 1025. Of course,any VOCs aside from the i-EBMs introduced into Stage 2 will also beconverted by this latter treatment into carbon dioxide and water, but,since these produces are not labeled with a non-ordinary isotope, suchas deuterium, the IR detector 1021 is easily tuned to provide distinctsignals based on e.g., deuterium content. Naturally, if in Stage 1 1010there has been a separation of compounds e.g., by chromatographic means(1013) the VOCs including i-EBMs are separated prior to introductioninto the IR Detector, whether catalytic combustion is utilized or not.The advantage of including catalytic combustion is that, rather thanneeding to utilize a tunable IR sensor, which tends to be complex andexpensive, a very simple and inexpensive IR sensor, tuned to detecte.g., deuterated carbon dioxide or deuterated water, may be utilized.

Shown in FIG. 20 is an example of a device according to this inventionwherein the treatment B of Stage 1 and the IR detector, treatment A ofStage 2 are arranged in series. According to this embodiment, the device1000 comprises a sample inlet 2000, which is directed to a three-wayvalve 2001. The three-way valve 2001 permits ambient air 2002 to passthrough an air scrubber 2003 to drive a sample of exhaled breath througha flow-through sample dryer/CO2 scrubber 2004 which removes endogenouswater and carbon dioxide from the sample, while allowing VOCs and i-EBMsto pass through. A heater, 2005, is associated with the flow-throughdryer/CO₂ scrubber to establish controlled temperature conditions, and,in the event that a VOC/i-EBM concentrator is also used, to inducethermal desorption from the concentrator at the desired time point.Where catalytic conversion of VOCs is utilized, a heater is provided togenerate elevated temperatures, although systems for conversion of VOCsto CO₂ and H₂O at about 50° C. are also available for this purpose. Onemerging from the flow-through dryer/CO2 scrubber 2004, the sample isdirected through another three-way valve 2006, which directs the samplethrough an IR detector 2007, and from there, via another three-way valve2008, via carrier pump 2009, to a vent 2010. In this embodiment, use ofa tunable IR sensor may be required to distinguish between i-EBMs andother VOCs which do not contain non-ordinary isotopes.

In FIG. 21, there is shown another embodiment of the device 1000 of thisaspect of the invention according to which, per FIG. 19, Stage 1,treatment B, and Stage 2, treatment B, are arranged in series. Accordingto this embodiment of the device of the invention, the exhaled breathsample is passed through a separation means, preferably e.g., aseparation column such as an appropriate GC column 2018, and then into acatalytic combustion chamber 2022 prior to being passed into a detector2024. Alternatively, as shown by the dashed line 2023, the separationcolumn 2018 may be bypassed or simply not included in an embodimentaccording to this aspect of the invention, by directly connecting theoutlet from the concentrator 2012 directly to the inlet of the catalyticcombustion chamber 2022. Those skilled in the art will be able toconfigure other alternate embodiments, based on the disclosure andteaching provided herein, to fit particular needs and circumstances.According to this embodiment 1000 (either including or not including aseparation means such as the separation column 2018) of the invention asshown in this figure, the device of this invention is utilized byintroducing a sample of exhaled air into sample inlet 2000 and fromthere, the sample is passed via three-way valve 2001 and istrapped/concentrated in a thermally desorbable concentrator 2012—e.g., ahydrophobic column, (e.g., tenax) from which adsorbed molecules aredesorbed by activation of heating means 2005, e.g., a peltier device orheating coil wound around the thermally desorbable concentrator 2012.Sample pump 2013 provides pressure as needed to draw sample in viasample inlet 2001 and to vent 2010 as needed. Preferably, a fan 2014 isincluded to ensure efficient and even heating of the concentrator 2012and dissipation of heat from the device 1000. Once the sample isadsorbed to the concentrator 2012, a three-way valve 2015 at the distalend of the concentrator 2012 permits ambient air 2002 to pass through anair scrubber 2003 to thereby provide scrubbed ambient air via the threeway valve 2015 to the distal end of the concentrator 2012. On actuationof the heating element 2005, the sample is desorbed from the thermallydesorbable concentrator 2012, and is driven from the concentratorproximal end of the concentrator 2012 via three-way valve 2001 ontoseparation column 2018, (if included in the particular embodiment, ordirectly to the chamber 2022, as noted above), preferably a gaschromatographic column selected to separate molecules according to theirpartition coefficient (boiling temperature) relative to the mobile andstationary phases in the column 2018. Preferably, the column 2018 isheated to a controlled temperature by heater 2011 to achievereproducible molecular separation and retention times on the column2018. Sample molecules emerge from the distal end 2019 of the column2018 at characteristic retention times. In this embodiment of the deviceaccording to this invention, the sample stream is directed to enter acatalytic combustion chamber 2022 where any VOCs and i-EBMs areconverted to ordinary carbon dioxide and water, if arising fromendogenous VOCs or comprising non-ordinary isotopes, if arising fromi-EBMs. Any such molecules are then detected, at characteristicretention times, via IR detector, 2024 which, of course, detects thewater or carbon dioxide coming off the column, albeit at thecharacteristic retention times of the VOCs from which they have beengenerated. The IR detector, of course, distinguishes any water or carbondioxide thus generated depending on whether non-ordinary isotopes arepresent in the water and carbon dioxide, or not. The thus analyzedmolecules are then passed from the detector 2024 and then vented 2025.In this embodiment, the IR detector 2024 may be tuned to detect, e.g.,water or carbon dioxide containing non-ordinary isotopes (e.g., ofhydrogen, carbon or oxygen), while ignoring detection of carbon dioxideor water arising from catalytic combustion of endogenous VOCs which donot contain non-ordinary isotopes. Preferably, the detector 2024 doesnot require tuning and is set to detect the characteristic signal of aparticular non-ordinary isotope of interest (e.g., deuterium, carbon oroxygen).

As discussed above, in Stage 1, i-EBMs are collected from the breath andseparated from water, carbon dioxide and, optionally, other volatileorganic compounds (VOCs) that may interfere with the subsequentanalysis. In its most basic form (Stage 1-A), this may be accomplishedby simply passing a portion of the breath through a low pressurescrubber (e.g., Nafion tubing) prior to entering the detector. For lowerconcentrations of i-EBMs, the scrubber can be replaced with aconcentrator (Stage 1-B), and for samples containing multiple i-EBMs,chromatographic separation can be included (Stage 1-C). In Stage 2, thecaptured i-EBMs are detected, analyzed, and optionally quantitated by anappropriate sensor, such as an IR-based detector. For i-EBMs withcharacteristic or intense absorption bands, the parent molecule ismeasured directly (Stage 2-A). In cases where this is not true, it maybe necessary to convert the i-EBM into a more readily detected species.When an organic molecule is combusted, carbon atoms from the moleculeare oxidized into CO₂ and hydrogen atoms are oxidized to produce water.Isotopically-labeled organic compounds give rise to correspondingisotopically-labeled combustion products. For example, an isotopologueof acetone containing ¹³C in place of place of hydrogen would generate¹³CO₂ and D₂O, respectively. ¹³CO₂ and D₂O are readily measured IRactive species. Since CO₂ and water are typical combustion products forall organic molecules, combusting any suitably labeled i-EBM, regardlessof its inherent IR absorption, will result in the same IR-activeproducts. By tuning the IR detector to measure these common combustionproducts (e.g., ¹³CO₂ and D₂O) instead of the parent i-EBM, thecombination of a catalytic combustion chamber and an IR detectorfunctions as a “universal” detector for any i-EBM. In fact, the utilityof this embodiment of the device is not limited to medication adherencemonitoring applications. Any VOCs may be analyzed in this way, therebyproviding significant added flexibility by providing a universal VOCdetector.

The various elements depicted in FIG. 19 can be combined to produceseveral distinct devices. The most basic of these devices is produced byjoining the Stage 1-A and Stage 2-A paths in series. Alternatively, themost complex design combines paths 1-C and 2-B, see FIG. 21.

In this embodiment of the device, there is provided a novel detector ofcompounds comprising non-ordinary but preferably non-radioactive (i.e.stable) isotopes, i.e. i-EBMs. With respect to the key application ofinterest here, the device is a novel medication adherence device basedon measurements of, for example, cold isotopologues of water in humanbreath. Stable cold isotopologues of water include, but are not limitedto: 1) H₂ ¹⁸O, 2) H₂ ¹⁶O, 3) H¹⁶OD, 4) D₂ ¹⁶O, and 5) H¹⁸OD, and/orstable cold isotopologues of carbon dioxide. Appropriate routes of drugadministration include but are not limited to: oral, intravenous (IV),transdermal, rectal, vaginal. A key point of novelty in this device isthat the water and CO₂ being used to detect medication adherence are NOTbeing generated by the body, but rather by mechanisms within the sensor.The system allows a common detection algorithm to be used to detect agreat many different drugs, markers, VOCs and the like. For medicationadherence monitoring, different types of EDIMs (Exhaled Drug IngestionMarkers) may be used alone or in combination. In a preferred embodiment,the device according to this invention exploits the use of awell-developed cold isotopic monitoring systems for water and/or CO2 formany types of i-EDIMs/i-AEMs/i-EBMs and can function with or without theuse of a baseline breath sample. A baseline breath sample can be used tosubtract off any background VOCs in the breath, e.g., DHO and D₂O, butthe baseline levels for these compounds are likely so low that nobaseline breath sample may be needed.

The detection can be accomplished with or without a mini-gaschromatograph (mGC). Without using the mGC, there is little delay whichis required in an mGC-based process, and results can be obtained in verynearly real time with time otherwise required for separation therebyeliminated. That is, in this embodiment, after the breath is sampledonto the tenax trap, the trap temperature is rapidly increased to about180° C. to desorb the VOCs from the trap and into the catalyticcombustion stage. Depending on the particular VOCs to be analyzed, anoptimal contact time with the catalyst to efficiently convert the i-EDIMto D₂O is selected. The D₂O then passes into the IR cell, where it maytake a few seconds to be analyzed (typically 16-64 IR scans are run forreliable statistics). This whole process may take between 30 seconds to1 minute.

This mode of analysis is limited to detecting only an integrated mass ofDHO and D₂O. With an mGC, the system can separate compounds based onboiling points prior to entering an IR detector or alternate detectorelement. Where IR is used, this may be used in a manner similar to MOSused in an existing mGC-MOS configuration. It permits robust detectionof DHO and D₂O in breath to identify many different types of deuteratedor other non-ordinary isotope containing i-EDIMs (i-AEM or metabolitesof i-AEMs).

In connection with this aspect of the invention, it is noted thatPicarro, Inc., provides a Micro-Combustion Module (A0214) to removeinterfering organics from water samples, in-line and efficiently. Thatmodule is disclosed as able to: improve data quality for water isotopeanalysis, treat samples in-line to decompose interfering organics;integrate seamlessly with Picarro's A0211 High-Precision Vaporizer, andto deploy effortlessly in the lab or the field—minimal footprint andenergy requirements. Designed to eliminate organic interferences fromwater isotope analysis using a fully in-line process, and installedbetween Picarro's High-Precision Vaporizer and the Picarro L2130-i waterisotope analyzer, the Micro-Combustion Module (MCM) is described asproviding seamless operation by passing a gaseous phase sample from thevaporizer over an enclosed element. The resulting oxidation convertsorganics into minute quantities of carbon dioxide and nascent water. TheMCM includes a self-contained micro-reactor element that can be easilyreplaced in the field. The MCM effectively removes spectral interferencefor commonly occurring alcohols and plant products includingmulticomponent mixtures of alcohols, terpenes and green leaf volatiles.It has optimal efficacy for samples containing total organics inconcentrations typical for many plant extracts (<0.5%) due to theproduction of nascent water. Higher concentrations of alcohols, such asthose found in certain beverages, will not be completely broken down inthe MCM. However, the process is highly reproducible and can createhigh-precision fingerprint data. This Picarro module may be incorporatedinto the Type II device according to this invention.

6.2.6 Detectors and Separators

a. CMOS, IR and Mass Spec Detection of Compounds with IncludedNon-Ordinary Isotopes Such as Deuterium

Use of IR sensor technology enables use of deuterated and othernon-ordinary isotope containing markers. Depending on the size of thegas sampling cell, detection of deuterated breath markers at levelsabove 1000 ppbv are readily detected. In the past, gas phase IRtechnology has typically not been able to go much below 1000 ppbv unlessa large, multi-pass gas cell or a molecule that has a huge IR absorptionis used. This is rapidly changing, however, and new solutions areconstantly being developed in this field.

A Nicolet 6700 FTIR, for example, has a detection limit of around 1 ppmfor acetone/IPA/deuterated acetone/deuterated IPA using a 5-L gassampling cell. Inclusion of a concentrator (such as that disclosedherein above in connection with the mGC), 1-L of breath is concentrateddown to a volume of about 1-10 cc. This decreases detection limits toenable detection of EDIMs in breath after pill ingestion. A diodelaser-based IR instrument is preferred for detection as they emit a muchhigher intensity light versus continuous light sources (e.g., the ETCEverglo source used in the Nicolet 6700 FTIR). Using such a modificationprovides detection limits 10-100 times lower than in the unmodifieddevice. Near InfraRed (NIR), Mid Infrared (mIR), diode laser, FTIR,Cavity Ring Down Spectroscopy (CRDS), and related systems are availablecommercially, for example, from Daylight Solutions, Inc., San Diego,Calif.; Picarro, Inc., Santa Clara, Calif., and the like. Picarro, Inc.,for example, affirms its sensors to measure in the low (e.g. 10) partsper trillion range for particular analytes. Organic compounds (bothnormal and deuterated) can be analyzed by infrared (IR) spectroscopy forboth qualitative and quantitative purposes. Either a FTIR (fouriertransform infrared) spectrometer can be used to continuously monitor theentire mid-IR wavelength range (4000-400 cm⁻¹ or 2.5-25 μm) or a tunablelaser diode with an IR detector can be used to monitor selectedwavelengths within this range (for example 4.3, 6.8, 8.3, 9.1 and 10.8μm laser diodes available from Daylight Solutions. A laser diode-basedIR spectrometer can also be used in a cavity ringdown mode (CRDS) tomonitor the IR absorption of a gas as a time-based measurement insteadof an intensity-based absorption measurement used in FTIR spectrometry.The advantages of this configuration is the very high sensitivity andprecision of the measurement, resulting in much lower detection limits.See also U.S. Pat. No. 8,410,560, and patent publication no. US2012/0267532. In a further embodiment, particularly utilizing evolvingsolutions for “mass spec on a chip”, compounds evolved in the exhaledbreath are subject to mass spec analysis, either in a central facilityor, preferably, built into the SMART® analytical device. Appropriatemass spec technologies appropriate for use in this application include,for example, that reported by Cheung et al., “Chip-Scale Quadrupole MassFilters for Portable Mass Spectrometry”, J. of MicroelectromechanicalSystems, V.19, Issue 3, pp. 469-483, (2010), and the M908 deviceavailable from 908 Devices, Inc., 27 Drydock Ave., 7th Floor, Boston,Mass. 02210, and U.S. Pat. Nos. 8,816,272; 8,525,111; and 8,921,774.

b. Miniature Gas Chromatography—mGC

In one embodiment according to this invention, the SMART® devicecomprises a miniature gas chromatograph, or mGC. According to thisembodiment of the invention, volatile organic compounds (VOCs) in theexhaled breath of subjects is introduced into a portable mGC devicewhich separates the VOCs according to partition coefficients for theVOCs as between a mobile phase and a stationary phase inside the mGCcolumn. For purposes of the present invention methods, known in the artcan be brought to bear for this purpose. Thus, see, for example,Andrews, A. R. J., Z. Wu, and A. Zlatkis. “The separation of hydrogenand deuterium homologues by inclusion gas chromatography,”Chromatographia 34.9-10 (1992): 457-460. Such systems may include, butare not limited to, for example, Sigma Aldrich b-Dex 110 Product No.24302, 60 m×0.25 mm i.d. 0.25 mm film thickness; b-Dex 110 Product No.24301, 30 m×0.25 mm i.d. 0.25 mm film thickness; CD Type: b (beta)Derivative: Dimethyl Phase: Non-bonded; 10%2,3-di-O-methyl-6-0-TBDMS-b-cyclodextrin embedded in SPB-35 poly(35%phenyl/65% dimethylsiloxane) (intermediate polarity phase); SigmaAldrich b-Dex 325, Product No. 24308, 30 m×0.25 mm i.d. 0.25 mm; CDType: b (beta) Derivative: Dimethyl Phase: Non-bonded; 25%2,3-di-O-methyl-6-O-TBDMS-b-cyclodextrin embedded in SPB-poly(20%phenyl/80% dimethylsiloxane), (intermediate polarity phase), and thelike. Results obtained using such systems can be seen, for example, inGas Chromatographic Determination of Isotopic Molecules by means of OpenTubular Thick Layer Graphitized Carbon Black Columns (J. Chromatog. 34(1968) 96) (utilizing a custom made column: 9.6 meters, 0.15 mm I.D.open tubular thick layer graphitized carbon black column, 87.5° C.Further information on such products is available, for example, fromRestek at restek.com.

6.3 Detailed Description of a Third Embodiment (Type III) of theImproved Smart® Device:

The Type III device according to this invention is a much simplifieddevice for medication adherence monitoring. According to this embodimentof the device, components of the Type I device as described above insection 6.1 are included, while others are dispensed with. Thus,preferably, as described above, the Type III device may, but does notnecessarily, include exhaled breath capture and concentration. Wherethis is not included, exhaled breath is directly exposed to sensors. Inthe Type III device, compound separation is not required asdiscrimination is achieved at the level of compound detection. Accordingto this aspect of the invention, at least two sensors are utilized: —Onespecific to the EBM or i-EBM and one sensitive to other VOCs. Bydifference, the concentration of the EBM of interest is calculated byon-board logic. An example of a device which could be adapted for usefor this purpose according to this embodiment has been described in theliterature, for a completely different purpose, by Toyooka et al., J.Breath Res. 7 (2013), “A prototype portable breath acetone analyzer formonitoring fat loss”. According to that report, acetone contained inexhaled breath is identified as a metabolic product of the breakdown ofbody fat and is expected to be a good indicator of fat-burning. Theynote that while, typically, gas chromatography or mass spectrometry areused to measure low-concentration compounds in breath, they state that“such large instruments are not suitable for daily use by diet-consciouspeople”. Naturally, the Type I and Type II embodiments of the SMART®device according to this invention as described herein provides justsuch a device, and, in addition to MAM applications, those devices maybe well applied for the metabolic monitoring purposes of concern toToyooka at al. Nonetheless, Toyooka et al., describe a prototypeportable breath acetone analyzer that has two types ofsemiconductor-based gas sensors with different sensitivitycharacteristics, enabling the acetone concentration to be calculatedwhile taking into account the presence of ethanol, hydrogen, andhumidity. To investigate the accuracy of their prototype and its use indiet support, they conducted experiments on healthy adult volunteers inwhich they found that breath acetone concentrations obtained from theirprototype and from gas chromatography showed a strong correlation.Moreover, body fat in subjects with a controlled caloric intake andtaking exercise decreased significantly, whereas breath acetoneconcentrations in those subjects increased significantly. They concludedthat their prototype is practical and useful for self-monitoring offat-burning at home or outside to help prevent and alleviate obesity anddiabetes. The device described by Toyooka et al., included a pressuresensor to detect exhaled breath and used a first gas sensor with“particularly high sensitivity to acetone”, (platinum-doped tungstenoxide, Itami, Japan), and a second sensor which has “almost equalsensitivity to both acetone and interference gases such as hydrogen andethanol” (tin oxide, SB-30, FIS, Inc.). The sensors were operated at 400deg. C., and differential calculations of output from the two sensorswas used to determine the acetone increases and decreases in exhaledbreath on different activities by subjects.

For purposes of a Type III device according to the present invention, acommercial embodiment of a platinum-doped, tungsten oxide sensor isproduced and utilized for acetone-specific detection where an AEM ori-AEM which generates breath acetone elevations (e.g., using isopropanolas the AEM) is used. Naturally, the first sensor may be selected foralternate EBM specificity than for acetone. A second sensor, such as thetin-oxide SB-sensor, is utilized in combination to measure othercompounds in the exhaled breath, to enable acetone (or other EBM)specific calculations to be achieved. Such an embodiment of a Type IIIdevice using dual-MOS sensors, of course, affords only 2-dimensions ofselectivity (i.e. the “array” coatings and the signal processing used toreject interference signals and deduce the acetone concentrations fromthe two signals). To achieve enhanced reliability in medicationadherence monitoring contexts, a concentrator as described above in theType I and Type II SMART® device is included ahead of the dual MOSarray, thereby providing 4-dimensions of selectivity and sensitivity,(concentrator sorbent, desorption temperature, array coating and signalprocessing), a significant enhancement over the device described byToyooka et al. The included concentrator protects the “naked” MOSdetectors from environmental contaminants and would therefore alsogreatly improve longevity. The concentrator would separate humidity,hydrogen, carbon dioxide, carbon monoxide, methane and othercontaminants from the e.g., acetone signal. The Type III SMART® deviceaccording to this aspect of the invention would preferably beapproximately “cigarette-pack” sized.

In an preferred embodiment of the Type III SMART® device according tothe invention, all components of the Type I device as described aboveare included and are incorporated here by reference, except that theseparation means, e.g., the mGC, is excluded, and the sensor accordingto this embodiment of the device are dual sensors with differentialsensitivities to analytes to enable detection and measurement ofspecific analyte(s) of interest.

In light of the forgoing disclosure in this section, it will beappreciated that the device (or a system incorporating the device)according to this invention for medication adherence monitoringcomprises;

-   -   a. an exhaled breath sampling module which obtains a sample of        exhaled breath from a subject;    -   b. an exhaled breath analysis module operatively coupled to the        breath sampling module so as to receive from the breath sampling        module a sufficient quantity or fraction of the sample of        exhaled breath to permit analysis of the constituent components        of the exhaled breath sample or fraction of the exhaled breath        sample; and    -   c. an exhaled breath kinetics module for determining kinetics of        appearance and disappearance of a marker identified by analysis        of the constituent components of the exhaled breath by the        exhaled breath analysis module.

It will be appreciated that the exhaled breath sampling module, exhaledbreath analysis module and exhaled breath kinetics module arepreferably, but not necessarily all included in a unitary, portabledevice.

In addition to using experimental approaches as disclosed and enabledherein above and in the examples, those skilled in the art will notethat sophisticated software, such as WinNonlin, can be used to model andpredict intra-individual and inter-individual variability of key PKparameters (Pharsight Corporation, Mountain View, Calif.).

Those skilled in the art will also appreciate that analysis of measuredexhaled breath components is optionally conducted on a central datarepository after EDIM concentration-time data is uploaded/transmittedfrom the portable device, or it is conducted locally on the SMART®device itself.

Those skilled in the art will also understand from this disclosure that,although this patent disclosure discloses and enables use ofcompartmental PK analyses (see Example 28), the invention is alsooperative using non-compartmental PK approaches, as described, forexample, by implementing WinNonlin software (Pharsight Corporation,Mountain View, Calif.).

Accordingly, the invention includes a device or system wherein theexhaled breath kinetics module calculates, for a given marker identifiedby analysis of the constituent components of an exhaled breath sample ofa subject obtained at a time t₁, whether the concentration of the markeris consistent with the expected concentration of the marker at the giventime t₁. This is done with reference to stored pharmacokineticparameters from the subject for the given marker and the dosage interval(T), if the subject had been adherent to a set regimen for introductionof the marker or a precursor of the marker into the subject over adefined time period prior to obtention of the exhaled breath sample.

Likewise, in an alternate embodiment, the device (or systemincorporating the device) according to this invention, the exhaledbreath kinetics module calculates, for a given marker identified byanalysis of the constituent components of an exhaled breath sample of asubject obtained at a time t₁, whether the concentration of the markeris consistent with the expected concentration of the marker at a timet₁, with reference to stored pharmacokinetic parameters obtained from alarge population of subjects for the marker and the dosage interval (T),if the subject had been adherent to a set regimen for introduction ofthe marker or a precursor of the marker into the subject over a definedtime period prior to obtention of the exhaled breath sample.

An optimized device or system according to this invention is optimizedby including in the device:

-   A. a sensor selected for accurate detection in the exhaled breath of    at least one subject of at least one Exhaled Drug Ingestion Marker    X, EDIM_(x) produced on ingestion of at least one Adherence Enabling    Marker, AEM_(x);-   B. data storage (as in hard drive, flash drive, EEPROM, in a form    now known or which is developed in the future) operatively coupled    to the sensor, for retention of data generated by the sensor in the    course of characterizing the pharmacokinetics of the EDIM_(x) in the    exhaled breath of a subject, Y, or in a population of subjects, Z;    and-   C. computing means, (including, for example, a programmed central    processing unit) which compares each such measurement for each    subject or population of subjects with stored data, as described    herein below, for said subject or population of subjects, preferably    in real time or near real time. For each measurement of the    concentration of EDIMx, a measure of adherence A is generated by the    computing means for each subject.

The characterizing data for storage preferably includes measurementdata, to within defined confidence limits, of:

-   -   a. the Limit of Detection (LoD) of a sensor included in said        device for said marker;    -   b. the background level of said marker or interferents in said        subject or population of subjects;    -   c. the half life of appearance (t_(1/2a)) and elimination        (t_(1/2e)) of said marker from the exhaled breath of said        subject or population of subjects;    -   d. the steady state concentration of said marker in the exhaled        breath at various time points during Adherence Enabling Marker        (AEM) dosing, selected from the group consisting of trough        (C_(Trough,SS)), maximum (C_(MAX,SS)), and other time point post        dosing of the AEM concentrations of said subject or population        of subjects; and    -   e. the time required to attain the maximum concentration        (T_(MAX)) of said marker from the exhaled breath of said subject        or population of subjects.

Such a device according to this invention is preferably configured tointegrate the pharmacokinetic parameters defined above to provide anadherence lookback window, T_(AdhWindow), defined as the period of timerequired for the marker (EDIM) concentration in breath of the subject todecay from an initial value (C_(EDIMo)) to a lower concentration(C_(EDIM,Limit))

$T_{AdhWindow} = {\frac{t_{{1/2}e}}{0.693}*{\ln \left( \frac{C_{EDIMo}}{C_{EDIMLimit}} \right)}}$

wherein:

C_(EDIMo)=original or starting concentration of marker (EDIM) in breathat times equal to or greater than T_(MAX) (i.e., C_(EDIMo)≦C_(MAX)) ofsaid patient;

C_(EDIMLimit)=the final concentration of EDIM in breath of said patient,provided that, if C_(EDIMLimit) denotes the limit of EDIM detection dueto the device LoD or background interference, it would define themaximum T_(AdhWindow); and t_(1/2e)=the elimination half life for saidEDIM.

Such a device preferably exhibits a T_(AdhWindow) between about 1 hourand about 400 hours, and includes a sensor with a LoD for the marker ofbetween 1 part per trillion and 5 parts per billion. In one preferredembodiment, the sensor is adapted to distinguish between ordinary andnon-ordinary isotopes present in EDIMs and volatile compounds whichotherwise would interfere with selective measurement of EDIMs in theexhaled breath.

7.0 IMPROVED SMART® COMPOSITION OF MATTER AND METHODS OF MAKING AND USETHEREOF

Depending on the mode of SMART® medication adherence monitoring, (e.g.,AMAM, IMAMA, CMAM), and the embodiment of SMART® device in use (Type I,II, or III), an appropriately matched SMART® composition is employed. Insection 7.1 below, we describe AEMs and compositions of mattercomprising AEMs which are adapted for use in a SMART® system whichincludes the Type I embodiment of the SMART® device according to thisinvention. In Section 7.2 below, we describe i-AEMs and compositions ofmatter comprising i-AEMs which are adapted for use in an i-SMART systemwhich includes the Type II embodiment of the SMART® device according tothis invention.

7.1 Detailed Description of a First Embodiment of the Improved Smart®Composition of Matter:

In developing the present invention, commercial imperatives relevant tomanufacture of SODFs containing volatile marker molecules (AEMs) havebeen carefully considered, experimented with and optimized to achieveexcellent methods for making and containing the AEM formulation, anddeployment with APIs. 2-butanol is utilized herein as a non-limitingexemplary marker (AEM) for SMART® system adherence monitoring. While2-butanol was disclosed in WO2013/040494 as a marker, the compositionsof matter disclosed herein provide advancements in the art by resolvingsuch matters as flashpoint of volatile AEMs during formulation and softgel encapsulation of the marker, acceptability of the AEM to subjectsreceiving administered medication, and by disclosing a combination ofmarker and excipients which optimize handling and/or processing of themarker composition, encapsulation properties, and improving tolerabilityand acceptability of the marker(s) when included in API dosage forms.

7.1.1 the AEM Composition According to this Embodiment of the Invention

Within this disclosure, considerable disclosure and attention is focusedaround use of 2-butanol or isopropanol (IPA) as Adherence EnablingMarkers (AEM), for generation of Exhaled Drug Ingestion Markers (EDIMs)(which, in the case of 2-butanol as the AEM is the ketone, 2-butanone,as the EDIM and in the case of IPA as the AEM, the EDIM is acetone),which is/are detected in the exhaled breath following ingestion ofmedication, those skilled in the art will appreciate that other AEMs andEDIMs may be used for this purpose, as disclosed, for example, inWO2013/040494. In addition, while the present disclosure focuses onspecific excipients and combinations thereof with the AEMs disclosedherein, those skilled in the art will appreciate that other equivalentexcipients and AEMs may be utilized.

In a first AEM composition according to this invention, at least orexclusively the following key components are contained within a “soft”gelatin capsule:

a. An AEM, primarily exemplified herein by 2-butanol;

b. An optional flavorant, primarily exemplified herein by DL-menthol,vanillin, or combinations thereof;

c. An optional bulking agent, primarily exemplified herein bypolyethylene glycol. It will be appreciated by those skilled in the artthat, in general, pharmaceutical grades of all materials mentionedshould be used for utilization in human dosage forms.

In a first AEM composition according to this invention, only the AEMitself (e.g., 2-butanol or IPA or combinations thereof) is included in agelatin capsule which is then combined with or administered concurrentlywith an API for medication adherence monitoring. In a second AEMcomposition according to this invention, the AEM is combined with one ormore additional components, including but not limited to flavorants,bulking agents, other excipients, or the like, as described above.

As noted above, those skilled in the art will appreciate that AEMs otherthan 2-butanol or IPA may be appropriate for a particular applicationand can, based on the disclosure and guidance provided herein, makeappropriate modifications to the formulation to accommodate alternateAEMs, volumes, concentrations and chemical interactions. Flashpointconsiderations with respect to the AEM, if it is a volatile compoundsuch as 2-butanol, define parameters for consideration in the safehandling of medication fill formulations in commercial contexts. Workingtemperatures above 25 degrees centigrade using compounds with a 22degree centigrade flashpoint, for example, are less than optimal. Theflashpoint of neat 2-butanol is about 22 degrees centigrade. However, bycareful experimentation with different amounts of 2-butanol, and carefulselection of the amount and nature of flavorants, bulking agents, andother excipients optionally included in the AEM formulation, we havebeen able to increase the flashpoint of the 2-butanol formulation suchthat the effective flashpoint of the 2-butanol is increasedsignificantly to greater than 26 degrees centigrade. Surprisingly, wehave found that certain combinations of vanillin, DL-menthol, or both,as disclosed herein, increase the 2-butanol flashpoint. Likewise,careful selection of bulking agent also can have this desirable effect.

The particular combination of DL-menthol and vanillin has been found inour preliminary testing to be well tolerated by subjects receiving theAEM formulation (see Examples below), but, of course, other combinationsof different flavorants (or no flavorant) could likewise be accommodatedand adapted for use according to this invention. In addition, as notedabove, the combination of flavorants with the volatile AEM has thesignificant advantage of raising the flashpoint of the volatile AEM.

With respect to the bulking agent, this has several important functions.First, the bulking agent is utilized to bring the total volume of theformulation to a desired total volume. For a consistent volume to befilled in each soft-gel capsule, it is important for the total volume tonot be too small for the relevant commercial fill operation, otherwiseundue errors are introduced into the total concentration of AEM betweendifferent capsules. Those skilled in the art know how to calculatevolumes for particular fills which will eliminate or reduce this aspectof variance such that essentially no statistically significant variancein EDIM measurement on the breath can be attributed to differences inAEM fill volumes used in the soft-gel capsules. Second, the bulkingagent is preferably one which does not retard release of the AEM upondissolution of the capsule containing the AEM. Third, preferably, thebulking agent itself is compatible with the containment material for theAEM such that integrity of the soft-gel is not interfered with by any ofthe constituents included in the AEM formulation. Typically, soft-gelcapsules include at least one or a combination of the followingcomponents: a shell forming composition, such as but not limited togelatin; a plasticizer, such as but not limited to glycerin, sorbitan,sorbitol, or similar low molecular weight polyols, and mixtures thereof.The art of soft gel capsule production is mature and those skilled inthe art will be aware of at least the following patent documents whichdisclose various compositions and methods of making this componentrelevant to the present invention: U.S. Pat. No. 5,641,512; U.S. Pat.No. 4,164,569; U.S. Pat. No. 8,241,665; U.S. Pat. No. 8,338,639. Thereare several well-known and respected commercial producers ofsoft-gelatin capsules, including, but not limited to, Patheon, 4721Emperor Blvd., Suite 200, Durham, N.C. 27703-8580, USA; Catalent PharmaSolutions, 14 Schoolhouse Road, Somerset N.J. 08873; LD Industries, 1725The Fairway, Jenkintown, Pa. 19046-1400; and Soft Gel Technologies,Inc., 6982 Bandini Blvd., Commerce, Calif. 90040, to name but a few.

In a preferred embodiment according to this invention, the AEMcomposition is contained within a soft-gel composition as follows:

Ingredient Use Gelatin Acid Bone (Type 195), NF, EP; clear Shell Polymergelatin with no colorants or opacifiers added Sorbitol/Glycerin blendPlasticizer

Thus, in one preferred formulation according to this invention, there isincluded:

Formulation A: 20 mg 2-butanol+0.7 mg DL-menthol+5 mg vanillin+9.3 mgPEG-400

Formulation B: 40 mg 2-butanol+1.4 mg DL-menthol+10 mg vanillin+18.6 mgPEG-400

It will be appreciated that there are many different grades ofpolyethylene glycol, PEG, and the selection of PEG-400 in the particularpreferred formulations mentioned above comes as a result of optimizationof the particular formulation to include the volatile AEM, 2-butanol,and the particular flavorants, DL-menthol and vanillin. The designationPEG-400, indicates an average molecular mass of 400 g/mole. Since thestructure for PEG is H—(O—CH₂—CH₂)_(n)—OH, for PEG-400, n=9. Of course,depending on the viscosity desired, PEG of different average molecularmass may be chosen as the bulking agent, with 3≦n≦50. PEG-400 isselected as a preferred component of the AEM formulation according tothis invention due to its combination of solubility, viscosity, andother characteristics. It is soluble in water, it acts as a solvent andcarrier for the 2-butanol, and flavorants and has a positive effect inincreasing the flashpoint of the formulation. We have explored use ofother grades of PEG, including, but not limited to PEG-200, PEG-600, andthe like. These grades of PEG are functional in the present invention,but we have found that he PEG-400 grade is optimal when the selected AEMis 2-butanol. PEG-400 and PEG-600 are both listed in the US FDA'slisting of Inactive Ingredients for approved drugs.

We have also found, via experimentation, that for the purposes ofdelivering the AEM, the ratios of the AEM (e.g., 2-butanol), to PEG-400,to flavorants, is also important. Thus, as can be seen by comparing theabove Formulation A to formulation B, the ratios of these components isretained when twice the amount of AEM is included in the formulation.Naturally, those skilled in the art will appreciate that there can besome variation in these ratios without loss of the ability tosuccessfully deliver the AEM and measure the EDIM on the exhaled breath.However, the ratio disclosed herein has been found to be preferred,providing miscibility of the marker in the formulation, stability intemperature cycling and chilling studies, room temperature stability,and dispersion in 0.01 1N HCl and neutral buffered solution. Theformulation, in addition, can be scaled to produce GMP batches forclinical trials and commercial use, it releases rapidly and reliably inthe stomach, and is anticipated to exhibit long-term stability 1-2 yearshelf life at room temperature), while, at the same time, permittingencapsulation in the smallest possible size (i.e. less than 6 mm or lessthan 5 mm or smaller, if possible) of soft gel capsule (thereby takingup the minimum amount of volume to permit API filling of capsules andother SODF's containing the AEM-soft gel formulation). It is alsopreferred that the gel capsule thickness containing the AEM be as thinas possible where AMAM is desired to be achieved. A softgel containingthe AEM (whether 2-butanol alone or in combination with otherexcipients) is provided.

For early testing, each formulation is placed directly (i.e. withoutencapsulation of the AEM in a soft gelatin capsule) in a white size 4LiCaps® hard gelatin capsule and sealed. The sealed white size 4 LiCaps®capsule will then be placed in a white size 0 LiCaps® capsule which isNOT sealed.

For delivery of an API, the AEM formulation is preferably included in asoft-gel capsule which is then included in a solid dosage form includingthe API, in a format such as was disclosed in WO2013/040494 but improvedas disclosed herein. In a preferred embodiment, the soft-gel capsulecomprising the AEM formulation according to this invention is introducedinto the apical half of a hard gelatin capsule. The lower portion of thecapsule is filled with API composition, and the capsule is closed,thereby containing both the AEM soft-gel capsule and the API in a singledosage form.

7.1.2 the AEM Composition According to this Embodiment of the Inventionand its Method of Manufacture

The art of encapsulation of solids and liquids is an advanced art area.However, the requirements of the present invention include:

-   -   a. the need to prevent loss of the volatile AEM (e.g.,        2-butanol, IPA) in the course of encapsulation or storage;    -   b. the need to prevent migration of the AEM into the API        compartment;    -   c. the need for rapid release of the AEM so that rapid        documentation of medication adherence can be achieved by        detection of the EBM in the exhaled breath.

These objectives are achieved for this aspect of the invention byproducing soft gelatin capsules containing the AEM. Depending on thedesired mode of MAM, the capsule containing the AEM is optimized forrapid, intermediate or slow dissolution in the biological system. Thus,for AMAM, extremely thin wall thickness is preferred (see below) so thatappearance of the EBM in the exhaled breath is not unduly delayed.Typical hard gel capsules, such as LiCaps® capsule and Conisnaps®capsules are approximately 0.11 mm thick (Capsugel, Morristown, N.J.),whereas softgel capsules typically have a wall thickness of 0.64-0.76 mm(Catalent, Somerset, N.J.). For IMAM and CMAM, these considerations maybe less critical, and, in fact, appropriate retardants to dissolutionmay be utilized to extend the time from which a medication is taken tothe time that adherence has to be confirmed using an appropriateembodiment of the SMART® device according to this invention.

For the production of a soft gelatin encapsulated AEM according to thisinvention, those skilled in the art will appreciate that soft gelatincapsule technology is based on hermetically sealing a liquid in agelatin shell. It is typically practiced using the rotary die method,although other manufacturing technologies exist.

In the rotary die method, 2 gelatin films are fed between a set of diescontaining pockets for forming the capsules. A wedge is used between thedies to inject the fill material between the ribbons such that it formsthe capsules in the die cavities as they rotate together. The 2 gelatinribbons are sealed using a combination of heat and pressure tohermetically encapsulate the fill material.

The gelatin formulation is selected based on the desired properties ofthe capsule and to be compatible with the fill. Typical gelatinencapsulation formulations include glycerin and/or sorbitol asplasticizers in ratios to gelatin between about 0.5:1 to 0.8:1.

The levels of plasticizer and thickness of the ribbon are adjusted toform capsules that are strong enough to withstand normal handling. Therelationship between plasticizer level, shell thickness, and capsulegeometry, to capsule strength and VOC permeability is intuitive, butalso highly interactive, i.e., changing one will often be additive orsubtractive with another.

When choosing a system for encapsulation of a VOC into a soft gelatincapsule, the following considerations come into play regarding VOC lossand capsule breakage.

Plasticizer: Low level=reduced VOC permeation, but increased brittleness(more likely to break)

Capsule Shell: Thick capsule shell=reduced VOC permeation and increasedcapsule strength (less likely to break), but may impact the rate ofrelease of the VOC. Typical shell thickness levels range from 0.025″ to0.040″, with no real constraint provided the tooling is optimized forthe thickness. Values outside of these levels are not typical and areoptimized for production of the soft gelatin capsules containing the AEMdisclosed and claimed herein.

The soft gelatin capsules according to this invention are made by mixingany excipients (including, but not limited to, bulking agents and/orflavorants), preferably under vacuum until all materials are dissolvedand then the AEM is added under positive pressure, preferably under ablanket of inert gas, such as, but not limited to, nitrogen. Theformulation is stored under an inert atmosphere and is utilized in theencapsulation procedure as described above, followed by drying andpackaging.

For purposes of this aspect of the invention, we have successfullyproduced a soft gelatin capsule containing the AEM (2-butanol) with awall thickness of as thick as 0.030″ and as thin as 0.020″. The thinnestwall thickness results in a faster release time.

This testing was conducted on “uncoated” softgels comparing formulationA (40 mg of 2-Butanol, 10 mg of Vanillin, 1.4 mg of Menthol and 18.6 mgof PEG400) with a 0.030″ wall thickness to Formulation B softgel wherewe removed the Menthol and Vanillin, raised the 2-Butanol to mg and thePEG400 to 20 mg with a softgel wall thickness of 0.020″. In in vivotesting an overall faster release as shown in FIG. 18j . A baselinebreath was obtained, then the softgel formulation A was swallowed andbreath samples were collected at 10, 20 & 30 minutes post ingestion. At60 minutes, a further baseline breath was obtained and formulation Bsoftgel was ingested, and breath samples were again collected at 10, 20and 30 minutes after this baseline breath.

We have confirmed the stability of 2-butanol within a soft gelatincapsule containing 40 mg 2-butanol; 18.6 mg PEG 400; 10 mg Vanillin; 1.4mg menthol under accelerated conditions (40° C.; 75% relative humidity)and in real time conditions (25° C.; 60% relative humidity). For bothconditions, we observe excellent stability and retention of the AEM insoft gelatin capsules. Three month stability under acceleratedconditions are considered to be predictive of twelve month stabilityunder real time conditions. See FIG. 18 i.

7.1.3 AEM Capsule Overcoating

As an alternative to increasing shell thickness to contain a VOC such asan AEM according to this invention, we have explored application of acoating to the capsule to reduce the permeation of the AEM from thecapsule. Surface coating methods may include, but are not limited to,spray coating, ink jet printing, thermal transfer, laser printing, dipcoating and the like.

Thus, where it is desired to rapidly release the AEM, and very thingelatin wall thicknesses are used to contain the AEM, soft gelatincapsules containing the AEM in a preferred embodiment are over-coated.Coatings for this purpose are known in the art, for example, by thetrade names SmartSeal, ProtectSeal, Opadry II, Opadry 200, SmartCoat,BASF Protect, and the like. Thus, Kollicoat Smartseal® 30 D is describedby its manufacturer, BASF, as a “unique solution in pellet and particlecoating, where other products are too tacky to be applied withoutindividual items sticking together. Kollicoat® Smartseal D featuresoutstanding taste-masking, ensures quick release of the activeingredients in the stomach and offers superior protection with a reducedamount of coating, resulting in lower costs and more efficientproduction processes”. OPADRY® 200, manufactured by Colorcon, Inc., iscoated in a 24″ fully perforated O'Hara Labcoat II coating pan. Per themanufacturer, 15 kg of biconvex placebo tablets (10 mm diameter) arecoated to a 4% weight gain (WG) with the same lot of a blue Opadry 200formulation.

In our studies with coated and uncoated gelatin capsules containing theAEM, (40 mg of 2-butanol+PEG 400 (18.6 mg)+vanillin (10 mg)+menthol (1.4mg) and shell thickness of ˜0.03″) we used 0.1 N HCl as disintegrationmedia and utilized disintegration criteria consistent with The UnitedStates Pharmacopeial Convention (2008, available athttp://www.usp.org/usp-nf/official-text/accelerated-revision-process/accelerated-revisions-history/disintegration-0)to obtain an average Disintegration Time (DT) for gelatin capsulescontaining the AEM. We explored several coatings (n=6 capsules for eachcondition tested). % weight increase is an indication of the averageamount of coating applied; Rupture Time (RT) is the average time beforeAEM odor could be detected; Disintegration Time (DT) is as noted above.Non coated gelatin capsules resulted in disintegration in about 4.7minutes; Opadry II coating: % weight increase 20, 14.5, 10.4, RT: 7.5-8minutes; DT: 13.6, 11.5 and 8.1 minutes respectively; Opadry 200: %weight increase 20, 14.6, 9.4, RT between and 8 minutes; DT: 16.2, 13.6,and 9.7 minutes respectively; SmartCoat 30D: % weight increase 20, 14.3,9.6, RT: 7.0-8.0, 5.5, and 4.5 minutes; DT: 8.2, 7.3, and 5.9 minutesrespectively; SmartSeal 30D (20%) coated with ProtectSeal (3%), RT:7.0-8.0 minutes, DT: 9.3 minutes; BASF Protect: % weight increase 15.5,9.3, 5.2, RT 9, 6.5, 4.5; DT: 11.9, 7.8, 5.9 minutes respectively. In aseparate set of studies, we enclosed three capsules, each containing 40mg AEM (2-butanol) for 16 hours in a sealed bag, and then sampled theheadspace. The coating type and results are shown in the table below,(relative to AEM detected in the headspace of sealed bags containinguncoated capsules, which is set as 100% and all other amounts arenormalized relative to the uncoated capsule headspace measurements):

2-Butanol release (% Un-coated) Coating Capsule Set 1 Capsule Set 2Capsule Set 3 Type Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2Uncoated 100 100 100 100 100 100 SmartSeal + 69.7 99.4 56.6 67.5 57.466.6 ProtectSeal Opadry II 34.2 44.5 33.2 35.3 24.1 18.2 Opadry 200 0.20.9 0.8 0.4 0.9 0.4

As between softgel capsules of 0.02″ and 0.03″ thickness, we havemeasured butanol egress overnight and found no significant difference.In addition, with 0.03″ thick softgel capsules, as between 15%Opadry®200 and 20%, there was no statistically significant difference inegress. Thus, it appears that a 0.02″ thickness softgel capsule coatedwith 15% Opadry® 200 is a preferred embodiment. Finally, for accelerated6, month stability data (representing 24 months standard/real-time)conditions, a total egress from 0.03″ softgels, a total egress of about8% (e.g. 3.2 mg/40 mg), but, based on overnight egress data usingOpadry® 200, it appears that egress is reduced by a factor ofapproximately 100 (i.e. for either 0.02″ or 0.03″ thickness softgels).

From this, we conclude that AEM encapsulated in a thin gelatin softgelcapsule overcoated with an appropriate coating, e.g., Opadry 200,exhibits containment and release characteristics desirable for deliveryof AEM in combination with an API of interest. The API itself ispreferably, and typically is, contained in its own protective coating,including when delivered in a unitary dosage form with the AEM containedas described herein. Utilizing the gelatin capsule contained AEM asdisclosed herein, in combination with a wide array of APIs may beconducted according to procedures and structures disclosed inWO2013/040494.

It should further be noted in connection with this aspect of theinvention that, (in addition to Medication Adherence Monitoring (MAM),whether for acute, intermediate or chronic applications (AMAM, IMAM, orCMAM)), because this system is exquisitely adept at detection ofdissolution of dosage forms in the digestive tract, an additionalutility for this invention (device, system) is a method and compositionsfor measuring residence times and digestive activity. Compositionscomprising an AEM and a coating or coatings known to be resistant orsusceptible to dissolution in different compartments of the digestivetract are thus considered to come within the scope of this invention.Thus, a composition comprising an AEM encapsulated, for example, in asoft gelatin capsule and coated with a coating resistant to gastricdissolution provides a system for measurement of the rate at which aparticular individual or population releases a medication beyond thegastric chamber.

An enteric coating, for example, is a polymer barrier applied on oralmedication to protect drugs from the pH (i.e. acidity) of the stomach.Most enteric coatings work by presenting a surface that is stable at thehighly acidic pH found in the stomach, but breaks down rapidly at a lessacidic (relatively more basic) pH. For example, they will not dissolvein the acidic juices of the stomach (pH˜3), but they will in thealkaline (pH 7-9) environment present in the small intestine. Materialsused for enteric coatings include fatty acids, waxes, shellac, plastics,and plant fibers.

7.1.4 Additional Containment Methods and Formulations for Retention andDelivery of the AEM

Whether the AEM according to this invention is an “ordinary AEM” ascompared with an i-AEM, it is desirable to ensure the AEM is not lost inthe formulation process, and is stable when co-packaged/formulated withan API of interest. In WO2013/040494, for example, HPC(hydroxypropylcellulose, a well-known excipient in the pharmaceuticalarts) was suggested for inclusion with volatile markers in capsules toprovide stable compositions for long-term storage in hard gel capsuleswith minimal hydroscopic forces. It was suggested that HPC “ties up”hydrogen bonding of 2-butanol, which in turn reduces its ability toattract water from the hard gel matrix that would dehydrate the hard gelcapsule and reduce its performance. Likewise, in WO/2013/038271, HPC wassuggested for inclusion in fill formulations as a polymer such that afill component (2-butanol, isopropanol, other VOCs) which, in theabsence of the at least one polymer will migrate into or through acapsule shell. Such methodologies may likewise be included for the AEMused in the system according to the present invention.

Furthermore, we have found it possible to produce a flowable dextrinpowder which contains significant amounts of AEM. Reference for thispurpose may be had to a series of patents to General Foods Corporation,including U.S. Pat. Nos. 3,795,747; 3,821,433; 3,956,508; 3,956,509;3,956,511, each of which describes alcohol (30-60% ethanol) containingdextrin powder and methods to make such powder. Such powders were stablewhen hermetically packaged. We have explored whether the AEM accordingto this invention may be formulated in a similar fashion. Usingmaltodextrin (commercially available, for example, as Maltrin M700 fromGPC, Grain Processing Corporation) we successfully achieved binding andretention of 2-butanol as a flowable powder (40.0% 2-butanol, 4.4% waterand 55.6% Maltrin M700. Left open to the atmosphere, the AEM-dextrinpowder retained from 36% 2-butanol at the time of formulation to about20% of 2-butanol after 48 hours. The dextrin readily dissolves in waterand in the digestive system, rapidly releasing the bound AEM.Accordingly, further aspect of this invention comprises the provision ofan AEM-dextrin formulation. In a first embodiment of a medicationaccording to this aspect of the invention, the AEM-dextrin powder isincluded in a hard gelatin capsule with an AEM. In a second embodiment,the AEM-dextrin powder is encapsulated in a soft gelatin capsule, asdescribed above in sections 7.1.2. In a further embodiment, theAEM-powder is coated, as in and section 7.1.3 above. In yet a furtherembodiment, the AEM-powder is included in a gelatin capsule which isthen coated, as in section 7.1.3. In another embodiment, a polymericstarch based sugar bead is impregnated with liquid 2-butanol or likeAEM, and optionally but preferably, coated with a PVA or similarmaterial to trap the 2-butanol or like AEM in the sugar bead. Thisfinished “powder” is utilized in a capsule with the active drug,converted to a slurry for surface coating of a medication, or otherwiseassociated with an active pharmaceutical ingredient to produce a SMART®formulation for use according to the present disclosure. In a furtherembodiment, as described in the examples below, a stable metal carbonateof a preferred alcohol marker, e.g. 2-butanol, isopropanol, or the likeprimary or secondary alcohol, is converted to a carbonate, includingcarbonates which include non-ordinary but stable isotopes, which can bepowderized and applied to the surface of an API by a device andtechnology known in the industry, such as is available from NordsonCorporation, 28601 Clemens Road, Westlake, Ohio 44145. These procedures,embodiments and formulations are likewise applicable to i-AEMs (section7.2 below). FIG. 84 shows different strategies for associating the AEMwith an AEM and the resultant rate of EBM release. FIG. 84A shows acapsule formed with 72 mg 2-Butanol in Maltodextrin (4:1 w:w) in size 0LiCap top, with size 1

LiCap bottom seal. As can be seen, this results in peak 2-butanol in theexhaled breath within about 10 minutes of ingestion. FIG. 84B shows acapsule formed with 64 mg 2-Butanol in Maltodextrin (4:1 w:w), in size 4LiCap (interior coated with OPAGLOS) inside size 1 LiCap. As can beseen, this strategy results in a peak 2-butanol in the breath withinabout 30 minutes of ingestion. FIG. 84C succinctly shows how thesensitivity and rate of EBM release is dependent on theconfiguration/strategy used to deliver the AEM, e.g. 2-butanol, to thestomach. It is clearly related to the total thickness of the gelatinbarriers it has to traverse before being released into the gastricenvironment. Note the far left curve (designated as reference) is thefastest when only one gel barrier (2-butanol solution placed directlyinto a hard gel capsule). With a surface coating containing the AEM,e.g. via a powder, only one gel barrier has to be crossed before beingreleases into the stomach. In any case, it is anticipated that such anapproach results in considerably faster release and generation of theEBM than occurs with softgel in a hardgel capsule assembly. FIG. 84Dprovides another example of maltodextrin powder, “fluffed up” tooptimize loading with 2=butanol, to produce a freely flowable powder at40% loading by weight with 2-butanol. This was ingested (equivalent of40 mg 2-butanol powder mass) simply placed inside a hard gelatin capsule(Size o LiCap). The breath kinetics: 1st derviative response of the mGCto 2-butanone in breath vs breath sampling time. These examples show howrapidly the powder can release the 2-butanol in the stomach, relativeto, e.g. a softgel-based strategy. Each of these strategies permits abalance to be achieved between rapid release in the stomach versusacceptable stability and segregation when the AEM is packaged with anAPI. Spray drying, microemulsion, and microencapsulation oroverencapsulation technologies are advanced and permit an appropriatebalance to be struck between these competing requirements.

7.2 Detailed Description of a Second Embodiment of the Improved Smart®Composition of Matter—Compositions and Methods of Making and Use ofi-AEMs

Much of the enabling description provided in section 7.1 above forprovision of AEMs with APIs is applicable here, where delivery and useof i-AEMs is described in detail. However, because the background ofi-EBMs is so low, the mass of i-AEM that needs to be delivered accordingto this aspect of the invention to achieve AMAM, IMAM, and CMAM isgenerally much lower than when regular AEMs (i.e. AEMs that do notcontain non-ordinary isotopes) are utilized. Thus, whereas milligramquantities of 2-butanol may be required to achieve readily measurablequantities of 2-butanone in the exhaled breath shortly after delivery ofthe medication, microgram quantities of e.g., deuterated 2-butanol orisopropanol are all that is required to achieve detectable quantities ofdeuterated 2-butanone, or deuterated acetone. Because the quantities ofi-AEMs that need to be delivered are much reduced as compared withregular AEMS, the i-AEMs may be much more simply associated with, forexample, Solid Oral Dosage Forms (“SODFs”). For example, microdots ofi-AEMs which are entirely contained in rapidly dissolvable barriers maybe adhered to the exterior of existing SODFs. Alternatively, capsuleswhich are already imprinted with adequate quantities of an i-AEM, eitheron an external or an internal surface thereof, and adequately containedin a barrier, or included in a capsule shell compartment, are filledwith an API. Alternatively, inks comprising an appropriate i-AEM may beused to print on an existing SODF, with an over-coat spray of a rapidlydissolvable barrier being sufficient to contain loss of the i-AEM. In afurther preferred embodiment of a medication according to thisinvention, there is provided an AEM which comprises either or both (a) anon-ordinary isotope; (b) butanol, isopropanol, or both, either or bothof which may include a non-ordinary isotope, or other selected secondaryalcohols, or other AEMs. In a further embodiment, the medicationincludes a surface coating comprising an i-AEM. Given the sensitivity ofa D₂O detector described herein, a low quantity (1-10 mg) of adeuterated AEM placed on the surface of SODFs (solid tablets, capsules)is adequate to permit medication adherence monitoring. Surface coatingand containment, for example, in a blister pack or equivalent preservesthe i-AEM on the surface of the SODF.

In addition to simplifying means for delivery of the i-AEM, because ofthe low background level of i-EBMs in the breath, the period of timefollowing dosage that the i-EBM is unequivocally detectable in theexhaled breath can be extended well beyond the dose-by-dose monitoringshortly after each dose is taken/administered (AMAM), which has been thestandard paradigm for medication adherence monitoring to date. Use ofi-AEMs enables IMAM and CMAM, often many hours or even days followingadministration/taking of a given dose or multiple doses.

As shown in Table 1 below, the use of isotopic labeling for this medicalapplication has multiple advantages, including generating distinctmolecular entities in the sense of detection, but, in general, thesemolecules are not so altered as to give rise to regulatory concern; see,for example, “Guidance for Industry, Investigational New DrugApplications (INDs)—Determining Whether Human Research Studies Can BeConducted Without an IND”, U.S. Department of Health and Human ServicesFood and Drug Administration Center for Drug Evaluation and Research(CDER) Center for Biologics Evaluation and Research (CBER) October 2010Clinical/Medical, Section V (lines 292-323) for guidance on the use of“cold” (e.g., deuterium) isotopes in clinical trials, which indicatesthe low level of scrutiny for this type of isotopic marker from a USRegulatory Agency perspective.

Non-radioactive isotopes include a number of elements (e.g., H, C, O, N,S), but for a variety of reasons deuterium is one of the most promisingfor our adherence application, particularly when mid-IR (mIR) techniquesare contemplated to detect the i-EBM (see below Table). Accordingly,reference to deuterium herein, or any other specific isotope, is notintended to be limiting or to exclude the use of other stable(non-radioactive) isotopes.

Medical Isotope Stable, Non-radioactive Radioactive, Unstable Hydrogen¹H (protium) - 99.985% ³H (tritium) ²H (deuterium) = ²H - 0.015% Carbon¹²C - 98.89% ¹⁴C ¹³C - 1.11% Oxygen ¹⁶O - 99.759% ¹⁵O ¹⁷O - 0.037% [MRIscans] ¹⁹O ¹⁸O - 0.204% [PET scans] Nitrogen ¹⁴N - 99.63% No convenient¹⁵N - 0.37% [biochemical tracers] Sulfur ³²S - 95.00% ³⁵S(other S-based³³S - 0.76% radioisotopes very ³⁴S - 4.22% short lived) ³⁰S - 0.014%

The table shows examples of stable and non-stable isotopes that may haveapplications in biology (medicine), including application to humanbreath. For purposes of the present invention, it is the stable,non-radioactive isotopes shown in this table that are of principalinterest. Using isotopic labels in breath analysis has many advantagesincluding but not limited to 1) creating a distinctive “fingerprint” inthe breath, which can be used to distinguish labeled compounds fromendogenous compounds already present in the body from natural metabolismor diet (e.g., ingestion of food, flavoring additives, drugs orexcipients of drugs) and 2) can produce changes in the detectioncharacteristics (e.g., shifts in the absorption spectra using FTIR) thatmake these molecules easily distinguishable from major analyticalinterferants in biological media. The % data indicate the percent of allatoms of that particular element in this isotopic form.

Successful integration of isotopically labeled GRAS taggants into oronto hard gel capsules, pills, tablets, creams, topical compositions,vaginal compositions or rectal compositions for medication adherencewill have the following requirements (referencing deuterium as apreferred but non-exclusive isotope for this purpose): 1) an adequatemass of e.g., deuterated taggant be interfaced (e.g., be part of the APIitself, or be part of a taggant included with the API, so that upondelivery of the API, there is concurrent delivery of the taggant) to theactive pharmaceutical ingredient (API) to generate a deuterated i-EDIM(i-EBM) breath signal, which can be measured with a portable sensor(e.g., midIR) to confirm medication adherence;

2) the deuterated taggant is rapidly released from hard gel capsules,soft gel capsules, tablets, or other dosage form in which the taggant isprovided, and, in turn, rapidly generates the deuterated i-EDIM (i-EBM);

3) the deuterated taggant must be interfaced to the commercial capsule,tablet or other dosage form in a manner that does not alter itsperformance characteristics;

4) the deuterated taggants must be linked to the commercial pill orother dosage form (or clinical trial material) containing the API in away that does not cause issues with API CMC or pharmacokinetics (PK:ADME) including bioavailability, and/or pharmacodynamics (PD); and

5) the taggant must create a deuterium-labeled i-EDIM (i-EBM) that iseasily detected by a portable sensor (i.e., mIR device) in a sensitiveand specific manner.

Those skilled in the art will readily determine, in consultation withappropriate regulatory bodies, whether potential additional regulatoryassays (e.g., toxicology on GRAS component of hard gel capsule;toxicology of API with hard gel capsule containing deuterated GRAStaggants) may be required.

7.2.1 Chemistry for i-AEMs and i-EBMS

In order to make a 1^(st) generation medication adherence device(without use of isotopic labeling strategies), a number of studies wereundertaken. Key results include: 1) identification of several classes ofGRAS food additives suitable for definitive adherence (i.e., as taggantsinterfaced to medications that generate appropriate EDIMs in exhaledbreath shortly after oral ingestion to document adherence); 2)demonstration that metabolites of taggants (EBMs, including EDIMs orEDEMs) were detectable in human breath using gas chromatography-massspectroscopy (GC-MS), mGC-MOS, or variations of such techniques, and hadkinetics (breath concentration-time relations) that were suitable fordefinitive MAMS; and 3) production of a portable miniature gaschromatography metal oxide sensor (mGC-MOS) prototype to detect EDIMs.It having now been demonstrated that MAMS is technologically feasible(chemistry+physiology+sensor—all work), the present invention provides amore advanced, refined, and flexible medication adherence system basedon isotopic labeling (e.g., deuterium) chemical approaches.

A. Candidate Taggants for Definitive Medication Adherence Using i-AEMs:

A number of regulatory databases exist that provide information aboutfood additives, flavorings and colorings that are legally found in orwhich can be added to food. GRAS taggants are preferably selected fromthose provided in the authoritative, proprietary Leffingwell &Associates (Canton, Ga.) “Flavor-Base 2007”. This listing is the world'smost extensive database on GRAS flavoring materials and food additives(4,085 listings). All compounds in the Flavor-2007 database containinformation from the relevant FDA and international regulatorydatabases. In all 1,603 esters, 926 alcohols, 222 aldehydes and 557ketones were initially identified as potential taggant candidates. Ofthese the esters and carbonate esters can be used to easily generate awide variety of corresponding alcohols and carboxylic acids.

In this embodiment, depending upon the ester, the i-EBM could be 1) anisotopically-labeled ester, 2) an isotopically-labeled alcohol derivedfrom the isotopically labeled ester, and/or 3) an isotopically-labeledacid derived from the isotopically-labeled ester. In addition, variouscombinations of isotopically-labeled esters and their associated labeledacids and/or labeled alcohols could be used to provide unique i-EBMsignatures in the breath. The type of substituents may be varied tosterically/electronically alter the susceptibility of the ester tohydrolysis, and will thus regulate the rate of appearance of ester-basedlabeled i-EBM(s). The physicochemical properties (e.g., physical state,volatility) of the ester will be a function of its substituents (Rgroups). By incorporating various isotopic labels (preferably deuterium)into various atomic sites of the esters, various i-EBMs (arising fromthe ester, acid and/or alcohol) containing one or more isotopic labelsis/are generated that fulfill the requirements of an effective MAMS.

The following criteria are relevant to selection of appropriate i-AEMs(and, indeed, to AEMs, unless specifically referenced to i-AEM selectioncriteria): 1) state of matter: solid versus liquid; 2) taste: absent orpresent (pleasant vs unpleasant); 3) physicochemical properties: boilingpoint, melting point, Henry's Law constant (K_(H)); 4) PK properties:ADME, including metabolism rates and routes (non-CYP-450 to avoidadverse drug reactions [ADRs]); 5) extensive safety data: stability,toxicological data such as permissible daily exposure (PDE) in humansand LD₅₀ values in various species (typically in the gms/kg range fororal administration); 6) minimal-to-no implications from a regulatoryperspective (no impact on CMC of API [study drug or FDA approved drug]or PK/PD of API); and 7) metabolism of taggant generates i-EBMs that areeasily detected by the mGC-MOS or mGC-mIR (e.g., i-EBM is detected bythe sensor and is neither a significant endogenous chemical nor widelygenerated via ingestion of different foods or medications).

Based on these considerations, a preferred set of fourteen compounds areprovided in Table 2.

TABLE 2 Potential LD₅₀ Oral GRAS BP K_(x) at 25° C. Rat Taggant CAS MFMW (° C.) Structure Chemical Structure (

C

/

C) (mg/kg) 2-propanol (isopropyl alcohol)

C

H

O 60.1  82 secondary alcohol

3111  6045 2-butanol

C

H

O 74.12  99 secondary alcohol

2478  6480 2-pentanol

C

H

O 88.15 118 secondary alcohol

1638  1470 ethyl acetate

C

H

O

88.11  77 primary ester

 164 10147 ethyl butyrate

C

H

O

116.2  120 primary ester

 68 13050 ethyl isobutyrate

C

H

O

116.2  113 primary ester

 *80 13000 hexyl acetate

C

H

O

144.2  169 primary ester

 *46 30147 isoaryl (isopentyl) acetate

C

H

O

130.2  142 primary ester

 50 16600 triethyl butyrate

C

H

O

130.2  102 primary ester

 117 >5000 methylisobutyrate

C

H

O 102.1   90 primary ester

 *79  5000 methyl propionate

C

H

O

88.11  79 primary ester

 147  5000 propyl acetate

C

H

O

102.1  102 primary ester

 116  9370 2-pentyl butyrate

C

H

O

158.2  186 secondary ester

unknown unknown 2-pentyl acetate

C

H

O

130.2  133 secondary ester

 *31 unknown Key physicochemical properties of fourteen taggants forMAMS; Key: CAS, chemistry abstract service code; MF, molecular formula;BP, boiling point; KH, Henry Law's constant at 25° C. (= ratio ofcencentration of taggant in liquid to gas: CL/CG); LD50, oral dose ofarticle that causes 50% mortality in rats; *, indicates mathematicalestimate; values obtained from Merck Index (12.3 CD Version), ChemIDplusAdvanced and ChemExper.com.

indicates data missing or illegible when filed

The list contains three 2° alcohols and eleven esters (nine 1°alcohol-based esters, plus two 2° alcohol-based esters). Secondaryesters and their corresponding 2° alcohols offer many advantages indefinitive adherence. For example, 133 esters were identified in thefood database having boiling points ranging from 30 to 320° C.,indicating the wide diversity available for a technology like mGC-MOS,mGC-midIR, which are essentially “boiling point” detectors. Aliphaticesters are rapidly hydrolyzed to their corresponding alcohol andaliphatic carboxylic acids by esterases, which could serve as i-EBMs.Tables 3 and 4 below show common alcohols and carboxylic acids,respectively, formed from esters.

TABLE 3A Preferred secondary and tertiary alcohols- those that are GRAScompounds

LD50

Oral Dir- at Rat ect Struc- 25° (mg/ LC50

Food

CAS ME NW BP ture C.

kg) Rat

 1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

indicates data missing or illegible when filed

TABLE 3B Illustrative Examples of Select Alcohols and TheirPhysiochemical Properties (data derived from Merck Index, 14th Editionand ChemExp [http://www.chemexper.com/]) CAS Code R′—OH Molec FormulaPhysical Name of Alcohol Molecular Weight Properites

CAS: 67-56-1 MF: CH₃O MW: 32.04216 BP: 64.7° C. MP: −97.6° C. VP: 230.8 

at 37° C.

CAS: 64-17-5 MF: C₂H₆O MW: 46.07 BP: 78.3° C. MP: −114.1° C. VP: 114.8 

at 37° C.

CAS: 71-23-8 MF: C₃H₈O MW: 60.10 BP: 97.2° C. MP: −127°C. VP: 43.5 

at 37° C.

CAS: 67-63-0 MF: C₃H₈O MW: 60.10 BP: 82.5° C. MP: −88.5°C. VP: 

 

at 20° C.

CAS: 71-36-3 MF: C₄H₁₀O MW: 74.12 BP: 117.6° C. MP: −90° C. VP: 14.65 

at 37° C.

CAS: 78-92-2 MF: C₄H₁₀O MW: 74.12 BP: 99.5° C. MP: −114.7° C. VP: 13 

at 20° C.

CAS: 78-83-1 MF: C₄H₁₀O MW: 74.12 BP: 102° C. MP: −108° C. VP: 8.8 

at 20° C.

CAS: 75-65-0 MF: C₄H₁₀O MW: 74.12 BP: 82.4° C. MP: 25.6° C. VP: 88.0 

at 37° C.

CAS: 137-32-6 MF: C₅H₁₂O MW: 88.15 BP: 128° C. MP: −70 ° C. VP: 2.9 

at 25° C.

CAS: 71-41-0 MF: C₅H₁₂O MW: 88.15 BP: 137.5° C. MP: −79° C. VP: 2 

at 20° C.

CAS: 123-51-3 MF: C₅H₁₂O MW: 88.15 BP: 130° C. MP: −117° C. VP: 3 

at 20° C.

CAS: 763-32-6 MF: C₅H₁₀O MW: 86.13 BP: 132° C. MP: N/A VP: N/A

CAS: 4516-90-9 MP: C₅H₁₀O MW: 86.13 BP: 122° C. MP: N/A VP: N/A

CAS: 111-27-3 MF: C₆H₁₄O MW: 102.18 BP: 156.4° C. MP: −45° C. VP: 0.9 

at 25° C.

indicates data missing or illegible when filed

CAS, chemistry abstract number; MF, molecular formula; MW, molecularweight; BP, boiling point; MP, melting point; VP, vapor pressure atspecified temperature

Shown in Table 3B are alcohols that are commonly generated via enzymaticdegradation of GRAS food additives/flavorants and/or FDA approved drugs(e.g., esterase mediated degradation of esters to their correspondingacids and alcohols). By incorporating various isotopic labels shown inTable 1 (e.g., and preferably, deuterium), into substrates that createthe various alcohols shown but not limited to the above table, or eveninto alcohols directly, various i-EBMs are generated that fulfill therequirements of an effective MAMS.

TABLE 4 Illustrative Examples of Carboxylic Acids and TheirPhysicochemical Properties (data derived from Merck Index, 14th Editionand ChemExp [http://www.chemexp.com/]) CAS Code Molec Formula Name ofCarboxylic Acid Molecular R-COOH Weight Physical Properties

 

CAS:

MF:

MW:

BP:

° C. MP:

° C. VP:

 

 at

° C.

 

CAS:

MF:

MW:

BP:

° C. MP:

° C. VP:

 

 at

° C.

 

CAS:

MF:

MW:

BP:

° C. MP:

° C. VP:

 

 at

° C.

 

CAS:

MF:

MW:

BP:

° C. MP:

° C. VP:

 

 at

° C.

 

CAS:

MF:

MW:

BP:

° C. MP:

° C. VP:

 

 at

° C.

 

CAS:

MF:

MW:

BP:

° C. MP:

° C. VP:

 

 at

° C.

 

CAS:

MF:

MW:

BP:

° C. MP:

° C. VP:

 

 at

° C.

 

CAS:

MF:

MW:

BP:

° C. MP:

° C. VP:

 

 at

° C.

 

CAS:

MF:

MW:

BP:

° C. MP:

° C. VP:

 

 at

° C.

 

CAS:

MF:

MW:

BP:

° C. MP:

° C. VP:

 

 at

° C.

 

CAS:

MF:

MW:

BP:

° C. MP:

° C. VP:

 

 at

° C.

 

CAS:

MF:

MW:

BP:

° C. MP:

° C. VP:

 

 at

° C.

indicates data missing or illegible when filed

CAS, chemistry abstract number; MF, molecular formula; MW, molecularweight; BP, boiling point; MP, melting point; VP, vapor pressure atspecified temperature

Shown in Table 4 are different carboxylic acids that are commonlygenerated via enzymatic degradation of GRAS food additives/flavorantsand/or FDA approved drugs (e.g., esterase mediated degradation of estersto their corresponding acids and alcohols). By incorporating variousisotopic labels shown in Table 1 (e.g., and preferably, deuterium), intosubstrates that create the various acids shown but not limited to theabove table, or even into acids directly, various i-EBMs are generatedthat fulfill the requirements of an effective MAMS.

Compared to carboxylic acids, alcohols are more suitable i-EBMs for avariety of reasons (e.g., carboxylic acids have poor [high] K_(H) values(=C_(L)/C_(G), liquid to gas phase concentration ratio that cause themto partition preferably in blood versus breath)). In the case of 1°alcohol-based aliphatic esters (1° esters) such as ethyl butyrate,esterases rapidly create a 1° alcohol (i.e., ethanol). For 2°alcohol-based aliphatic esters (2° esters) such as 2-pentyl butyrate,they are rapidly hydrolyzed to their corresponding 2° alcohol (i.e.,2-pentanol) by esterases, particularly by carboxylesterases (e.g.,β-esterase). The carbon that carries the hydroxyl (—OH) group of primary(1°), secondary (2°) and tertiary) (3° alcohols is attached to 1, 2, and3 alkyl groups, respectively. The 1° and 2° alcohols are primarilyconverted (oxidized) via alcohol dehydrogenase (ADH) to theircorresponding aldehydes and ketones, respectively. In contrast to 1° and2° alcohols, 3° alcohols, due to steric hindrance with ADH, are veryresistant to metabolism in humans and thus are not ideal for MAMS,unless a 3° alcohol-based ester liberated a 3° alcohol (e.g., tert-butylbutyrate→tert-butanol), which was used as the EDIM. The aldehydes arefurther metabolized by aldehyde dehydrogenase (ALDH), which oxidizes(dehydrogenates) them to their corresponding carboxylic acid. Incontrast, methyl ketones undergo α-hydroxylation (e.g., conversion of2-butanone [methyl ethyl ketone, MEK] to 3-hydroxy-2-butanone [acetoin]via CYP-2E1 and CYP-2B, or conversion of 2-pentanone [methyl propylketone, MPK] to 3-hydroxy-2-pentanone) and subsequent oxidation of theterminal methyl group to eventually yield corresponding ketocarboxylicacids. The ketoacids are intermediary metabolites (e.g., α-ketoacids)that undergo oxidative decarboxylation to yield CO₂ and simple aliphaticcarboxylic acids. The acids may be completely metabolized in the fattyacid pathway and citric acid cycle.

We have tested and confirmed that 2° alcohols (or even 2° esters thatgenerate 2° alcohols) are excellent taggants for definitive adherencemonitoring. The presence (and persistence) of their correspondingketones (EBMs) in exhaled breath represents definitive proof ofingestion of a medication containing 2° alcohols as taggants. Ingeneral, due to increased steric hindrance, 2° alcohols are less good assubstrates for ADH relative to 1° alcohols. Likewise, the enzymaticpathways to degrade alcohol-derived ketones appear less efficient thanthose for alcohol-derived aldehydes. Given the fact that 1) the gastricwall has a high concentration of ADH and alcohols (e.g., ethanol) areknown to be significantly absorbed through the stomach, and 2) alcoholsundergo extensive first pass metabolism via ADH in the liver afterabsorption from the GI tract, it should not be surprising that when2-butanol is ingested, 2-butanone levels appear very rapidly in thebreath, and its concentrations significantly exceeds those of 2-butanol(ketone:alcohol ratio: 2-butanone/2-butanol>>1). In contrast, when2-butanol is administered via non-oral routes (e.g., transdermal, mucousmembranes, intravenous, eye) to humans, the ketone: alcohol ratio isreversed (<1), relative to the value for oral administration, since thetwo above factors would not be operative. In addition, the availabilityof a wide variety of 2° alcohols provides a large number of taggantsavailable for definitive adherence monitoring. In keeping with ourhypothesis that 2° alcohols (vis-à-vis 1° alcohols) would generateketones that would persist in the body and have significant excretion bythe lung, diabetic patients readily excrete ketones during thepathophysiological condition of diabetic ketoacidosis (DKA). Ketonesgenerated from other sources (e.g., orally ingested 2° alcohols) wouldalso be excreted by the lung. Using the mGC-MOS, we have already shownthese endogenous DKA-related ketones are easily distinguished from theketones which would be generated from 2° esters or alcohols, including2-butanone and 2-pentanone.

Below is a summary of some key advantages and disadvantages of usingesters, 1° alcohols and 2° alcohols for definitive MAMS:

B. Advantages and Disadvantages of Using Various Chemical Classes ofCompounds as i-AEMs/i-EBMs

1. Esters

Advantages

-   -   Great variety of GRAS food additives    -   Esterases generate corresponding alcohol and carboxylic acid via        enzyme systems that are widely present in humans and not easily        saturable    -   Many exist in liquid and solid state forms    -   Relative to alcohols, many more choices for selecting solids    -   Great variety of favorable tastes    -   2° alcohol-based esters such as 2-pentyl butyrate are primarily        degraded by carboxylesterase to 2-pentanol and butyric acid:

Disadvantages

-   -   Greater mass of taggant required to be interfaced to API in        order to generate a fixed mass of EDIM (e.g., 2-butanone)    -   Some esters not optimal from a stability standpoint    -   1° alcohol-based esters as GRAS compounds are much more common        than 2° alcohol-based esters in food databases; these alcohols        generate aldehydes, which are not ideal EDIMs relative to        ketones derived from 2° alcohols

2. 1° Alcohols

Advantages

-   -   Much greater variety of GRAS food 1° alcohols relative to 2°        alcohols    -   Larger 1° alcohols via ADH generate aldehydes, particularly        those that are branched, which are better EDIMs (e.g., low K_(H)        values; distinct from endogenous compounds) than more simple 1°        alcohols, but have lower vapor pressures

Disadvantages

-   -   ADH forms aldehydes from 1° alcohols, which are generally not as        good EDIMs as ketones, particularly with the more simple 1°        alcohols    -   Many have classic alcohol taste; may require CMC architecture        approaches or addition of taste “maskers” to avoid    -   Disulfiram, a drug used to treat alcoholism that blocks the        action of aldehyde dehydrogenase, may interfere with the        degradation of corresponding aldehydes, and cause side effects;        this effect is expected to be clinically irrelevant due to the        small mass of alcohol (or its corresponding ester) required for        definitive MAMS    -   Ethanol consumption (via interaction with ADH) can theoretically        reduce the conversion of 1° alcohol taggant to its corresponding        aldehyde; this has not been found to be clinically significant        for a number of non-ethanol alcohols (excludes methanol)

Note: In addition of 1° alcohols, a number of critically importantCYP-450 metabolic reactions for pharmaceutical agents, via dealkylations(FIG. 26), generate various aldehydes (FIG. 27), include formaldehydevia desmethylation, acetaldehyde via desethylation, propionaldehyde viadespropylation, and butyraldehyde via desbutylation.

3. 2° Alcohols

Advantages

-   -   ADH generates ketones, which generally have more favorable        physicochemical and metabolism characteristics as EDIMs than do        aldehyde EBMs.    -   The ADH that generates ketones from 2° alcohols is not affected        by genetic polymorphisms, as is the case with the ADH that        generates aldehydes from 1° alcohols.    -   Disulfiram, an inhibitor of aldehyde dehydrogenase, will not        interfere with the degradation of ketones formed from 2°        alcohols (e.g., methyl ethyl ketone, derived from 2-butanol, is        converted to 3-hydroxy-2-butanone via CYP-2E1 and 2B).

Disadvantages

-   -   Many have classic alcohol (ethanol) taste; may require

CMC architecture approaches or addition of taste “maskers” to avoid thetaste of these compounds.

-   -   Fewer 2° alcohols, relative to 1° alcohols, are listed in GRAS        food databases    -   Fewer 2° alcohol-based esters are listed in GRAS food databases        (e.g., these would generate the 2° alcohol, and later a ketone)

To assist understanding of this invention by those reviewing this patentdisclosure, reference is now made to FIG. 22 herein, which shows themetabolic fate of selected ordinary isotope and non-ordinary isotopelabeled alcohols, aldehydes and carboxylic acids. In humans alcoholdehydrogenases are a group of dehydrogenase enzymes that catalyze theinterconversion between alcohols and aldehydes (or ketones). Theirprimary function is to degrade alcohols. The enzyme is contained withinthe gastric lining and in the liver. Aldehyde dehydrogenases are enzymesthat catalyze the oxidation (dehydrogenation) of a various aldehydes.Multiple forms exist at various locations in humans, including thecytosol, mitochondria and endoplasmic reticulum. They are classified inthe following manner: Class 1 (cytosolic), Class 2 (mitochondrial) andClass 3 (tumor and other isozymes). Panel B shows potential isotopiclabeling sites. *, indicates a deuterium (stable isotope) label butcould be other types as shown in Table 1. Likewise, multiple deuteratedlabels could be placed on the molecule or alternately a combination ofdifferent isotopic labels (H, C and/or O-based) could be used; †,indicates a carbon isotopic label (see Table 1). Note: In this scheme,where appropriate, other potential isotopic labels (Table 1) could beused including 170 and/or 180 for ordinary oxygen. Direct isotopiclabeling of alcohols, aldehydes and acids is possible and adds tochemical diversity for MAMs. For example, during alcohol oxidation, theoxygen atom remains with the alcohol. It may be seen as adehydrogenation of the alcohol i.e. only one hydrogen atom leaves thealpha carbon, and the molecule converts from alcohol to the carbonyl,which would be an aldehyde for a primary alcohol. Thus, if oxygen of aprimary alcohol was labeled, it is possible to efficiently monitor theformation of the corresponding aldehyde after oxidation.

7.2.2 Methods of Making and Use and Compositions for Different Routes ofSmart® Medication (Containing, e.g., Deuterated i-AEMS) Administration:

In a first embodiment according to this aspect of the invention, thereis disclosed a SMART® medication, and a method of making the SMART®medication (or a composition comprising the SMART® medication),comprising an Active Pharmaceutical Ingredient (API) in combination withan AEM, that is at least one non-toxic, preferably Generally Recognizedas Safe (GRAS) volatile organic compound (VOC), or incipiently volatileorganic compound (i.e. on introduction into or onto a subject, the AEMis exhaled or gives rise to a compound which is exhaled), preferably adirect food additive, wherein at least one atom thereof is anon-ordinary isotope, e.g., a hydrogen of said VOC is replaced with adeuterium atom, such that, on administration (ingestion, topicalapplication, or other means of delivery) of the medication comprisingthe deuterium-labeled AEM (e.g., the VOC or a metabolite thereofcomprising) the deuterium atom is entrained and is detectable in theexhaled breath as an i-EBM. In addition, disclosed herein are certainnovel APIs or compositions comprising APIs wherein the API itselfincludes the non-ordinary isotope and acts as the i-AEM or produces thei-EBM.

The various types of i-AEMs which produce i-EBMs detectable in thebreath are discussed above. Depending on the route of administration ofthe medication, different formulations and physical arrangement of theAPI and i-AEM are preferred, as discussed below:

7.2.3 Oral i-AEM Medications

A wide variety of oral dosage forms including AEMs are disclosed inWO2013/040494, published 21 Mar. 2013, entitled “SMART™ SOLID ORALDOSAGE FORMS”. A number of physical forms for delivery of activetherapeutic agents in combination with markers were disclosed. Whereverin that publication there is mention of AEMs, per the present invention,non-ordinary isotopes may be included in the AEMs to produce i-AEMs,such that, upon introduction into the biological system, there isproduced in the exhaled breath i-EBMs which may be monitored accordingto the present invention. The contents of WO2013/040494 are hereinincorporated by reference as if fully set forth herein, to describe andenable those skilled in the art to utilize the various dosage forms thatcould be used to include i-AEMs according to the present invention.

In a preferred embodiment according to this aspect of the invention, thei-AEM is contained within a barrier, which keeps the i-AEM separate fromany API being co-delivered. The barrier may be composed of gelatin orother containment mixture known in the art. Where a very small quantityof neat i-AEM is desired to be used, it may be printed onto or otherwiseadhered to an existing dosage form and under and/or overcoated with aquickly dissolving i-AEM impermeable layer. Coatings known in the artfor this purpose may be utilized. Thus, microcrystalline cellulose,hydroxypropyl methyl cellulose, other polymeric or non-polymericbarriers, and the like, such as disclosed in, for example, U.S. Pat. No.6,352,719; US2007/0212411; US2004/0110891 and the like may be utilizedfor this purpose.

7.2.4 Vaginal/Rectal i-AEM Medications

Generally, non-toxic, and preferably GRAS secondary and tertiaryalcohols with between three and up to eight carbon atoms, including atleast one non-ordinary isotope of hydrogen (i.e deuterium), carbon,oxygen or nitrogen, are useful for this purpose. Thus, for example, anyor each of the following compounds which include at least onenon-ordinary but stable (non-radioactive) isotope may be used accordingto this invention as an i-AEM for non-oral delivery of i-AEMs for use incombination with the i-SMART® system: isopropanol; 2-butanol;2-methyl-2-butanol; 2-pentanol; 3-pentanol, and the like. Preferredsecondary and tertiary alcohols are those that are GRAS compounds.

In addition, while the present disclosure focuses on specific excipientsand combinations thereof with the i-AEMs disclosed herein, those skilledin the art will appreciate that other equivalent excipients may beutilized with the disclosed i-AEMs.

An optimized i-AEM composition is disclosed herein which comprises atleast or exclusively the following key components, mixed either prior todelivery or at the site of delivery at an appropriate concentration witha vaginal or rectal gel or other appropriate medium known in the art orwhich hereafter comes to be known in the art:

a. An i-AEM, e.g., deuterated 2-butanol, deuterated IPA, (but which maybe any of the i-AEMs discussed herein;

b. A gel medium for delivery of the i-AEM and/or Active PharmaceuticalIngredient (API);

c. At least one API, unless the i-AEM is being delivered in a placebo.

Those skilled in the art can, based on the disclosure and guidanceprovided herein, make appropriate modifications to vaginal or rectaldelivery formulations to accommodate alternate i-AEMs, volumes,concentrations and chemical interactions. When delivering an i-AEM via avaginal or rectal route, particularly where an anti-HIV API is beingco-delivered with the i-AEM, it is critical to ensure that the amountand concentration of secondary or tertiary alcohol acting as the i-AEMbe so low as to avoid inflammatory responses known to be caused whenhigh concentrations and amounts of alcohol, e.g., ethanol, are deliveredvia these routes. This is because it is known that high concentrationsof alcohol when introduced into the vagina or rectum, while able tocross the cellular barrier, induce significant inflammation. Aside fromthe associated discomfort, this also reduces a critical natural barrierto infection—actually increasing the susceptibility to infection by, forexample, HIV.

Surprisingly, successful detection of i-EDEMs in exhaled breath isachieved following inclusion of as little as about 3 to 10 mg of e.g.,deuterated 2-butanol. These doses, especially when dissolved in standardvolumes of microbicide gel (typically 4 ml), are very unlikely to elicitany inflammatory response at the site of delivery. For example, when adose range of about 3 to 30 mg of deuterated 2-butanol or IPA isdelivered vaginally or rectally in an appropriate carrier medium, e.g.,tenofovir placebo gel (i.e. the same medium in which tenofovir isdelivered but with or without the active agent tenofovir) even morereliable detection of deuterated 2-butanol, 2-butanone or acetone in theexhaled breath is achieved in a time frame and concentration sufficientto definitively confirm product placement with a high level ofconfidence, and without induction of inflammation at the delivery site.While greater amounts of i-AEM could be delivered by this route withoutcausing inflammation, it is preferred to deliver no more than 100 mg ofi-AEM, and, most preferably, to deliver between about 0.003 to 30 mg,and, most preferably, to deliver between about 0.03 and 3 mg. Becausethere is so little background when using i-AEMs, it is generallypossible to achieve reliable adherence monitoring utilizing amounts ofthe i-AEM that otherwise would not be easily detectable in exhaledbreath.

The physiology of the vaginal lining includes a significant barrier todelivery and diffusion of i-AEMs and APIs, due to the thick, stratifiedsquamous epithelial lining. Nevertheless, the inventors herein are ableto successfully deliver i-AEMs via the vaginal route. Thus, rectaldelivery, where a single epithelial cell layer forms the surface of therectum, is assured. Compositions, means and devices for rectal deliveryinclude gels, as for vaginal delivery, and such dosage forms assuppositories, which may include the API in an appropriate suppositoryvehicle known in the art, with the i-AEM admixed therein or in aseparate suppository compartment, coating or the like.

In formulating the i-AEM according to this invention for vaginal orrectal delivery concurrently with an API, it is important to utilizegels, lubricants, vehicles, and the like for i-AEM/API delivery which donot enhance transmission of disease causing agents, such as HIV. Forexample, see Begay et al., “Identification of Personal Lubricants ThatCan Cause Rectal Epithelial Cell Damage and Enhance HIV Type 1Replication in Vitro”, AIDS Research and Human Retroviruses, Volume: 27Issue 9: Aug. 23, 2011, which found that many over-the-counter personallubricants damage epithelial linings and, in some cases, enhance HIV-1replication. The same or similar formulation as used for Tenofovirplacebo gel may be used with substitution of a small fraction of theglycerol with the preferred alcohol according to this invention. From achemical standpoint the alcohol substitutes very well for glycerol inthese systems, and ensures excellent compatibility and solubility ofeven higher doses of alcohols.

Different i-AEM's may be included in a single composition in order topermit differential kinetics of appearance in breath to be optimized.Thus, more complex i-AEMs (higher carbon atom content) generally exhibitlonger half life in the breath, whereas the smaller, simpler i-AEM's aremore quickly cleared from the breath. Understanding these kineticconsiderations will permit those skilled in the art, based on thepresent disclosure, to select different i-AEMs and combinations ofi-AEMs, in order to tailor detection kinetics in the breath formonitoring adherence with respect particular APIs and different modes ofclinical use. In addition, or alternatively, a mixture of different APIsin a delivery medium or substrate, wherein each API is associated with adifferent i-AEM, may be utilized, and thereby, delivery of each API maybe tracked by detection of distinct markers on the breath, even if/whena mixture is prepared for delivery of several different APIs/i-AEMs.

In one embodiment according to this invention, a gel composition usedcommercially for vaginal or rectal delivery of tenofovir is utilized.This gel comprises 0 (placebo), 0.2, 1, or 5% tenofovir (GileadSciences, Inc., Foster City, Calif.) in a gel containing purified water,edentate disodium, citric acid, glycerin, propylparaben, methylparaben,and hydroxycellulose adjusted to pH 4 to 5. (Published Ahead of Print 10Oct. 2011. 10.1128/AAC.00597-11. Antimicrob. Agents Chemother. 2012,56(1):103. DOI: Nuttall et al., Pharmacokinetics of Tenofovir followingIntravaginal and Intrarectal Administration of Tenofovir Gel to RhesusMacaques). It will be appreciated by those skilled in the art thatdifferent compositions known in the art may be used as thevehicle/substrate for vaginal or rectal delivery of the i-AEM and API.For example, those skilled in the art are referred to U.S. Pat. Nos.7,192,607; 7,935,710; 8,367,098 for disclosure on such substrates andprocedures known in the art.

Those skilled in the art will be aware that a wide range of differentAPIs may be delivered via the rectum or vagina in a wide range ofdelivery media and mechanisms. Thus, while the terms “microbicide” or“microbicidally active” are generically applied to APIs for delivery bythese routes, and while the intent is to include such compounds astenofovir, emtricitabine, or combinations thereof (e.g., tenofovirdisproxil fumarate, marketed by Gilead Sciences under the trade nameVIREAD®), emtricitabine, and combinations of emtricitabine andtenofovir, e.g., TRUVADA®), the term is also intended to include anyknown or hereafter discovered reverse transcriptase inhibitors, proteaseinhibitors, other mode-of-action antiretroviral APIs and, indeed, anyother API for which vaginal or rectal delivery is a known or desiredroute of medication administration (e.g., valium).

In a preferred embodiment according to this aspect of the invention, themicrobicidal composition according to this invention includes an i-AEMand the microbicidally active compound is selected from the groupconsisting of marketed or investigational antiretroviral drugs usedeither solely or in combination to treat HIV infection, selected fromthe group consisting of:

-   -   A. Nucleoside Reverse Transcriptase Inhibitors (NRTIs) abacavir,        abacavir sulfate, azidothymidine, didanosine, dideoxycytidine,        dideoxyinosine, emtricitabine, lamivudine, tenofovir disoproxil        fumarate, stavudine, zalcitabine, zidovudine;    -   B. Non-nucleoside Reverse Transcriptase Inhibitors (NNRTIs):        delavirdine, efavirenz, etravirine, nevirapine, rilpivirine;    -   C. Protease Inhibitors (PIs): amprenavir, atazanavir sulfate,        darunavir, fosamprenavir calcium, indinavir, lopinavir,        nelfinavir mesylate, ritonavir, saquinavir, saquinavir mesylate,        tipranavir;    -   D. Fusion Inhibitors: enfuvirtide;    -   E. Entry Inhibitors—CCR5 co-receptor antagonist: maraviroc;    -   F. HIV integrase strand transfer inhibitors: raltegravir; and    -   G. Combinations thereof.

Where there is any concern about potential negative impact of admixtureof an i-AEM according to this invention with an API for delivery via therectal, vaginal, or indeed, any other route (including oral), because ofstability considerations (e.g., shelf-life, interactions between the APIand the i-AEM and the like), desire to avoid modification ofcompositions that have already received regulatory approval in theabsence of the i-AEM, or other considerations, the present inventioncontemplates means for admixture of the i-AEM at the site of delivery.This is achieved, for example, by maintaining the microbicidally activecompound and the i-AEM in compartments in the drug delivery means suchthat they are not in contact with each other until delivered vaginallyor rectally. Accordingly, in one embodiment according to this aspect ofthe invention, the API and i-AEM are maintained, prior to delivery, inseparate barrels of a two-barreled syringe. Alternate arrangements andembodiments to achieve a similar result include, for example, byincluding the i-AEM in (a) a Luer-lock tip which fits over the deliverymeans, e.g., a syringe, for the API in substrate; (b) in a slip-tip,either coaxially located, eccentrically located, or elongated, as in acatheter tip, which fits over the delivery means, e.g., a syringe, forthe API in substrate. Naturally, those skilled in the art willappreciate that in commercial embodiments, such combinations of physicalmeans for keeping the i-AEM and API separate from each other may berefined and may appear less like syringes than as unitary deliverymeans, but the operative principles inherent in these non-exclusiveexamples are the same. In another embodiment according to this aspect ofthe invention, the i-AEM is maintained in a softgel capsule which isbroken on delivery, e.g., by impact with a plunger, pin or needle tip,or the like, thereby mixing the i-AEM with vehicle, microbicidallyactive compound or both, at the site of delivery. Likewise, the intactsoftgel containing the i-AEM could be delivered from the syringe alongwith the microbicidally active compound at the time of product use, andthe softgel dissolves in the warm environment of the vagina. In yetanother embodiment according to this aspect of the invention, the i-AEMis coated on a syringe applicator tip which admixes the i-AEM ondelivery of the vehicle and the microbicidally active compound. In yetanother embodiment according to this invention, the Chemistry,Manufacturing and Controls (CMC) of a medication is modified to directlyaccommodate the i-AEM. For example, for this approach, in the vehiclefor a vaginally or rectally administered API, where glycerin isgenerally a major component of the vehicle, a tiny amount of glycerin isreplaced with the i-AEM, such as deuterated 2-butanol or IPA. Yetanother means of delivery of the API and i-AEM may be via a vaginalring, or similar device. According to this embodiment of this aspect ofthe invention, a polymeric drug delivery device provides controlledrelease of drug and i-AEM for intravaginal delivery over an extendedperiod of time. The drug/i-AEM delivery device is inserted into thevagina and can provide contraceptive protection, microbicidalprotection, and delivery of the i-AEM. By inclusion of the i-AEM, andconfirming ongoing detection of i-EBM in the exhaled breath, clinicianscan be assured that the drug delivery device is working correctly andhas not been prematurely removed. For rectal delivery, of course, a gelor suppository device/composition is preferred. With respect to asuppository, the i-AEM may be admixed with the API and suppositoryvehicle, or the i-AEM may be in a separate compartment which isdissolved upon API/suppository delivery, thereby releasing the i-AEM fordetection in the breath or for metabolism to generate the i-EBM.

7.2.5 Transdermal i-AEM Medications

A wide variety of transdermal medications and formulations exist and anyof these may be used in combination with the i-AEMs as disclosed herein.

Of particular interest with respect to this invention are “ethosomes”,defined by N. A. Pratima and T Shailee, IJRPS 2(1) JANUARY-MARCH 2012“Ethosomes: A Novel Tool for Transdermal Drug Delivery”, as follows:“Ethosomes are the slight modification of well established drug carrierliposome. Ethosomes are lipid vesicles containing phospholipids, alcohol(ethanol and isopropyl alcohol) in relatively high concentration andwater. Ethosomes are soft vesicles made of phospholipids and ethanol (inhigher quantity) and water. The size range of ethosomes may vary fromtens of nanometers (nm) to microns (p) ethosomes permeate through theskin layers more rapidly and possess significantly higher transdermalflux.” See also, for example, “ETHOSOMES: A NOVEL TOOL FOR TRANSDERMALDRUG DELIVERY”, Rasheed et al., World Journal of PharmaceuticalResearch, Volume 1, Issue 2, 59-71. Review Article ISSN 2277-7105. Seealso U.S. Pat. Nos. 5,716,638 and 5,540,934. Due to the inclusion ofalcoholic or VOC constituents in ethosomes, delivery of APIs with ani-AEM according to this invention, to produce i-EBMs is a preferredembodiment according to this invention for purposes of adherence usingtransdermally delivered medications. For other modes and compositionsfor API/i-AEM delivery via the transdermal route, those skilled in theart are directed to consider, e.g., Malakar et al., “Development andEvaluation of Microemulsions for Transdermal Delivery of Insulin”, ISRNPharmaceutics, Volume 2011, Article ID 780150, 7 pages,doi:10.5402/2011/780150; Kalluri and Banga, Transdermal Delivery ofProteins, AAPS PharmSciTech, Vol. 12, No. 1, March 2011 (#2011), DOI:10.1208/s12249-011-9601-6; Lauren A. Trepanier, “TRANSDERMAL DRUGS: WHATDO WE KNOW?” See, also, for example, U.S. Pat. Nos. 6,946,144;5,597,796; 7,537,795; 7,220,427. Clearly, this is a sampling oftechniques and compositions for transdermal delivery, and this is a wellestablished field in which those skilled in the art are able to utilizewhat is disclosed herein to enable transdermal delivery of the i-AEMs asdisclosed herein.

7.2.6 Other i-AEM Medications and Modes of Delivery

Those skilled in the art will appreciate, based on the disclosureprovided herein, that i-AEMs may be delivered by other modes, including,but not limited to, intravenously, intramuscularly, intraperitoneallly,intranasally, inhalationally, intraoccularly, while still producingi-EBMs detectable in the exhaled breath. Naturally, kinetics of i-EBMproduction, half-life, and other relevant considerations will come intoplay and appropriate modifications of compositions and times for breathmonitoring will need to be adjusted accordingly. Thus, for example,certain products that are available commercially include compounds whichcould function as i-AEMs if they were to include a non-ordinary isotopeaccording to this invention. Thus, for example, commercially availableeyedrops include chlorobutanol, while certain cosmetics includephenylethanol. Chlorobutanol is an alcohol that acts by increasing lipidsolubility, and its antimicrobial activity is based on its ability tocross the bacterial lipid layer.

Chlorobutanol is a widely used, very effective preservative in manypharmaceuticals and cosmetic products, for example, injections,ointments, products for eyes, ears and nose, dental preparations, etc.It has antibacterial and antifungal properties. Chlorobutanol istypically used at a concentration of 0.5% where it lends long-termstability to multi-ingredient formulations. Phenylethanol is anantimicrobial, antiseptic, and disinfectant, which is used also as anaromatic essence and preservative in pharmaceutics and perfumery.Accordingly, inclusion of at least a fraction of the total chlorobutanolor phenylethanol which is deuterated in such products which alreadyinclude non-deuterated forms of these molecules, provides a means formedication adherence monitoring by detecting the appropriate i-EBMproduced in the breath.

7.2.7 Preferred Aspects of the i-AEM Medications and CompositionsAccording to this Embodiment of the Invention

Based on the foregoing disclosure, it will be appreciated that in oneaspect of this invention, a SMART® (Self Monitoring And ReportingTherapeutic) medication is provided for delivery and monitoring ofadherence in taking or administration of at least one ActivePharmaceutical Ingredient (API) by a subject. This medication comprises:

-   -   (a) An i-API fraction, wherein at least one atom of at least a        fraction of the API is a non-ordinary but stable isotope; or    -   (b) An i-AEM, an Adherence Enabling Marker comprising at least        one non-ordinary but stable isotope; or    -   (c) Both an i-API fraction and an i-AEM;

such that, on taking or administration of the medication by or to thesubject, an i-EBM, an Exhaled Breath Marker comprising at least onenon-ordinary but stable isotope, is produced in the exhaled breath ofthe subject. Preferably, the stable but non-ordinary isotope is selectedfrom the group consisting of deuterium, or a stable but non-ordinaryisotope of carbon, oxygen, nitrogen, or sulfur. Preferably, where notthe API itself, the i-AEM is selected from the group consisting ofsecondary and tertiary alcohols, and more preferably, the secondary ortertiary alcohol is a compound which is a Generally Recognized as Safe(GRAS) compound, or a direct food additive, or both.

The SMART® medication is preferably delivered in a dosage form selectedfrom the group consisting of: a solid oral dosage form, (SODF),intravenously, transdermally, vaginally, rectally, intranasally,intraocularly, intramuscularly, inhalationally.

For optimal use of the medication as described above, a SMART® device isprovided for detecting in a gas sample a molecule which is labeled witha non-ordinary isotope wherein the device comprises a means forstripping the gas sample of moisture and carbon dioxide, optionally acatalytic incinerator for converting the molecule to carbon dioxide andwater, such that: (a) the isotope from the i-AEM is included in thewater fraction, such that, following catalysis, isotopically labeledwater is quantitated in the gas sample; (b) the isotope from the i-AEMis included in the carbon dioxide fraction, such that, followingcatalysis, isotopically labeled carbon dioxide is quantitated in the gassample; or (c) both (a) and (b). Preferably, the device includes a meansfor separating i-EBMs in exhaled breath prior to catalysis anddetection. The system and method according to this aspect of theinvention includes a method for medication adherence monitoring whichcomprises providing a SMART® medication to a subject and measuring inthe exhaled breath of the subject at least one i-EBM utilizing a Type IISMART® device. The method preferably includes monitoring kinetics ofappearance of i-EBMs in the exhaled breath and, depending on theparticular i-AEMs used and the route of administration, determiningadherence characteristics for the given subject and medication.According to this embodiment of the invention, monitoring is conductedfrom immediately to one hour, from one hour to several hours, or fromseveral hours to several days after the SMART® medication is taken bythe subject.

The system for medication adherence monitoring according to this aspectof the invention comprises:

A. Providing to a subject a SMART® (Self Monitoring And ReportingTherapeutic) medication for delivery and monitoring of adherence intaking or administration of at least one Active PharmaceuticalIngredient (API) by a subject, comprising:

-   -   (a) An i-API fraction, wherein at least one atom of at least a        fraction of the API is a non-ordinary but stable isotope; or    -   (b) An i-AEM, an Adherence Enabling Marker comprising at least        one non-ordinary but stable isotope; or    -   (c) Both an i-API fraction and an i-AEM;

such that, on taking or administration of the medication by or to thesubject, an i-EBM, an Exhaled Breath Marker comprising at least onenon-ordinary but stable isotope, is produced in the exhaled breath ofthe subject; and B. Measuring in the exhaled breath of the subject ani-EBM utilizing a device which comprises a means for stripping theexhaled breath sample of moisture and carbon dioxide, optionally, acatalyst for converting the i-EBM to carbon dioxide and water, suchthat: (a) the isotope from the i-EBM is included in the water fraction,such that, following catalysis, isotopically labeled water isquantitated in the exhaled breath sample; (b) the isotope from the i-EBMis included in the carbon dioxide fraction, such that, followingcatalysis, isotopically labeled carbon dioxide is quantitated in theexhaled breath sample; or (c) both (a) and (b).

8.0 IMPROVED SMART® SYSTEM AND METHODS OF USE THEREOF

This section of the patent disclosure relies on the previous sections (6and 7) to combine particular embodiments of the SMART® device withparticular AEMs and compositions of AEMs in a system which achievesheretofore unachievable results in the areas of AMAM, IMAM and CMAM. Thecombinations of the improved method, device, and composition, asdescribed herein, provides a system for medication adherence monitoringcapable of exquisite sensitivity and flexibility, including in theprovision of options for “lookback periods” of short, intermediate andchronic medication adherence.

This patent disclosure enables novel and inventive methods, means andsystems for reliably measuring acute, cumulative, chronic, and evenrandomly timed medication adherence monitoring within particular timewindows relative to the time a SMART® medication is taken or should havebeen taken. This represents a significant step forward in the art inthat acute medication adherence monitoring known in the art can beanalogized to a single measurement of blood glucose concentrationtesting in a diabetic, as compared to the HbA1C test for glycosylatedhemoglobin, which provides an indication of glycemic control over apreceding time period. It furthermore significantly alleviates theburden on clinicians and subjects whose adherence is being monitored, bysubstantially expanding the period in which monitoring can reliably beconducted.

The medication adherence monitoring tools disclosed and enabled hereinprovide progressively greater technological capabilities that facilitatedefinitive measurement and monitoring of adherence on an acute (dose bydose), semi-chronic (1-2 days) and/or a chronic (preceding 3 to 14 days)basis with maximum patient convenience and system accuracy. The SMART®system can be used to monitor adherence to drugs delivered via virtuallyany route, including but not limited to oral, i.v., transcutaneous,transdermal, intra-rectal, vaginal, i.p., inhalational, etc. Oralmedications represent the biggest market segment and understandingadherence to oral drugs will have the greatest impact on improvingclinical trial and disease outcomes in the near future. Thus, the tablebelow is focused on adherence technologies that can be used toeffectively monitor ingestion of any medication delivered within a solidoral dosage form (SODF), including capsules, hard tablets, sublingual(SL), and orally disintegrating tablets (ODTs).

To emphasize the variety of contexts in which the present systemoperates and to outline how this is achieved, the following tableprovides a useful reference:

SMART ® Adherence System Type 1B: Surface Type II: mid- Type 1A: MetalOxide Sensor (MOS)- Acoustic Wave (SAW)- Infrared (mIR)- Feature ofSMART ® based sensor engine based sensor engine based sensor engineSensory Configuration mGC-MOS mGC-MOS Dual MOS SAW mGC-mIR Type ofadherence Acute (pill Acute and Semi- Acute and Semi- Acute (pill Acute(pill by pill), by pill) chronic (preceding chronic (preceding by pill)Semi-chronic (preceding 1-2 days) 1-2 days) 1-2 days), and Chronic(preceding 3-14 days) Preferred Adherence- One simple (low Two simple(low One simple (low One higher One cold isotopologues Enabling Markerboiling point) boiling point) boiling point) boiling point of simple(low boiling (AEM) direct food direct food direct food food flavorantpoint) direct food additive (e.g., additives (e.g., additive (e.g.,(e.g., methyl additives (e.g., 2° alcohols: 2° alcohols: 2° alcohol:salicylate) 2° alcohols: 2-butanol) 2-butanol and 2-propanol) deuterated2-butanol 2-propanol) or 2-propanol) Mass of AEM required 20-60 mg 10-30micrograms 1-10 milligrams Breath marker(s) One AEM Two AEM metabolites:One AEM AEM itself One AEM metabolite: detected by SMART ® metabolite:ketones (e.g., metabolite: (e.g., methyl Ketone (e.g., sensor ketone(e.g., 2-butanone and ketone (e.g., salicylate) deuterated 2-butanone)2-butanone) acetone) acetone) Preferred location Small capsule (e.g.,softgel or hardgel) placed inside Standard flavorant Layer (e.g., ink ofAEM a DB Cap along with a physically separated active co-formulated withlogo) sprayed on a pharmaceutical ingredient (API) API in theformulationsmall area of matrix of ODT or the surface of any SL tablets SODFcontaining the API Minimum time to reliably ≧20 min (soft gel-based AEMinside DB cap with API Immediate (<30 sec) ≧5 min detect breath markerphysically separate); 5-20 min within softgel-based smart drugPersistence of breath Minimum Minimum Minimum <5-10 min 60-90 min tomarker 60-90 min 60-90 min to 60-90 min to several Days a maximum amaximum 1-2 days 1-2 days Can use with multiple Yes drugs and/or drugdoses? Potential interferents Minor None None-to-Minor Minor None tofunction Number of breaths Preferably 2 breaths Only 1 breath requiredrequired Approximate Size H2 × W4 × H2 × W4 × Cigarette Pack CigarettePack iPhone size L6 inches L6 inches size size

This aspect of the present invention provides an improved method,system, compositions of matter and apparatus for medication adherencemonitoring which extends the window of time from medication ingestion totime for confirmation of medication adherence. This is achieved by (a)characterizing the kinetics of appearance and disappearance of ExhaledDrug Ingestion Markers (EDIMs) in the exhaled breath of subjectsreceiving medications which include selected Adherence Enabling Markers(AEMs). The AEMs may themselves be the EDIMs or may be converted to theEBM (including EDIMs or EDEMs) in vivo via metabolism of the AEM.

In certain embodiments according to the invention, a first AEM, AEM₁, isselected which provides the ability to confirm adherence on an acute,dose by dose basis, by virtue of rapid appearance in and disappearancefrom the exhaled breath of subjects, in combination with a second AEM,AEM₂, selected for its ability to confirm adherence over a longer timeframe. For such embodiments, simple alcohols, such as 2-butanol, areselected for AEM₁. Such markers are rapidly metabolized in vivo intosimple ketones. The half-life for detection of the ketones is typicallyon the order of minutes to several hours, but generally less than, say,5 hours. For AEM₂, in such embodiments, an AEM with a longer half-lifein exhaled breath is selected. Isopropyl alcohol, (IPA), for example, isconverted in vivo into acetone. As shown herein, the half-life ofacetone derived from IPA is on the order of about 6.5 hours. Byappropriately adjusting the frequency of medication adherencemonitoring, based on the AEMs in use, subjects' adherence to medicationregimens may be checked on a dose by dose basis, or less frequently,with a lookback period defined by the kinetic considerations relating tohalf life, steady state concentration, and background noise and limitsof detection criteria, as defined in further detail herein.

In a further embodiment according to the invention, only AEM₂, isincluded in the medication.

In a further embodiment according to the invention, an AEM is selectedwhich includes an non-radioactive, non-ordinary isotope, such that thelookback period may be significantly extended, due at least in part dueto lower or almost non-existent background, and enhanced detectioncapabilities of the sensor and separation device utilized to confirmadherence.

Accordingly, it is an object of this aspect of the invention to providea medication adherence monitoring method, system, composition of matterand apparatus, which enables acute (dose by dose) and more extended(over more than a single dose and over the course of more than a singleday) medication adherence monitoring.

It is a further object of this aspect of the invention to provide amedication adherence monitoring method, system, composition of matterand apparatus, which alleviates the need for subjects to provide exhaledbreath samples for medication adherence monitoring only within tightlydefined time limits after the time a medication containing the AEM hasbeen administered or taken by the subject.

8.1 AMAM

For Acute Medication Adherence Monitoring (AMAM), the system accordingto this invention comprises a SMART® device for use in combination withat least one ordinary AEM or an i-AEM formulated in such a way that on adose-by-dose basis, it can be definitively determined that the correctperson has taken the correct dosage of the correct medication at thecorrect time. This is achieved by combining a Type I, Type II or TypeIII device with an AEM delivered for example in a softgel capsule or,for example, printed on an existing dosage form (in the case of ani-AEM) concurrent with delivery of the particular medication dosagebeing monitored. Within minutes up to about one hour after taking themedication dose, the Type I-III device as described herein in sections6.1-6.3, delivers definitive AMAM data (identify of the person bybiometric capture, identity and concentration of EBM included in theexhaled breath) all within minutes of taking a particular medicationdosage. The AEM may, of course, be an AEM as described herein in section7.1, or it may be an i-AEM, as described herein in section 7.2. In thelatter case, the device is preferably a Type II SMART® device, asdescribed herein in section 6.2, and may be used to advantage includingwhere only dose-by-dose AMAM is required.

8.2 IMAM and CMAM Using Ordinary AEMS and i-AEMS

Essentially all of the elements to practice the method and use a SMART®system according to this invention are described herein above for use ofordinary AEMs (i.e. AEMs not containing non-ordinary isotopes). A Type Ior III device in combination with an AEM which has a long half-life forappearance of the EBM in the exhaled breath, or persistence of the EBMin the exhaled breath, is all that is required for IMAM and CMAM usingordinary AEMs. Achieving a steady-state of medication delivery with amedication comprising one or more AEMs has predictable effects forpurposes of EBM measurement in the exhaled breath. Deviations from thesteady state EBM concentration are detected, and the subject may bequeried or challenged with respect to adherence.

In moving the field from AMAM to IMAM to CMAM, the ability to measure amarker in breath accurately for progressively longer periods of time iskey. This can be accomplished in preliminary studies with a givenindividual or a population of individuals, and with a given AEM, todetermine the half life in breath. Once if population PK has beenestablished for a given AEM, that data may be stored on board, or usedin a remote location, to analyze adherence for a given subject, and apreliminary phase for the given subject is not required.

By way of example, 2-butanol is converted to 2-butanone within minutesof release of 2-butanol into the digestive system (i.e. followingrelease of encapsulants or any other barriers implemented forcontainment of the AEM). 2-Butanone has a relatively short half-life forappearance in the exhaled breath, and definitive medication adherenceusing 2-butanol alone is thus limited to a relatively short look-backperiod of a few minutes to, at most, several hours. Medication adherencethus would need to be confirmed in that relatively short time-window,and failure to test adherence in that time window means that such datamay be lost altogether, even if the subject was perfectly adherent intaking the medication. Using an AEM such as isopropanol provides alonger window for medication adherence monitoring. Elevations in basalacetone exhalation due to ingestion of IPA as the AEM can be measuredover at least one 6.5 hour half-life, or even two such half lives, butthis requires measurement of the delta, that is change in acetone inexhaled breath and interference by endogenous acetone exhalation quicklybecomes a confounding factor thereafter. Use of more complex AEMsprovide options for more extended medication adherence monitoring (IMAMand even CMAM). Further details on the pharmacokinetic/pharmacodynamicconsiderations (which includes data on the breath concentration—timerelationships for EBM development and clearance for any given AEM/EBM)relevant to IMAM and CMAM are provided below in connection with thediscussion of use of primarily i-AEMs, but much of that disclosureapplies to use of ordinary AEMs.

To extend the ability of the SMART® system into reliable IMAM and CMAM,it is preferred to utilize a medication adherence monitoring system andmethod which comprises providing an i-SMART® medication or compositionof matter, as described above (section 7.2), to a subject and using thedevice, as described above (section 6.2), to detect and quantitate anon-ordinary isotope in the exhaled breath of the subject. In apreferred embodiment, the method is applied to medication adherencemonitoring. However, for the avoidance of doubt, any device or method orsystem which utilizes a novel device as disclosed herein is includedwithin the scope of this invention, including when in a field or utilityunrelated to medication adherence monitoring.

Because of the very low background of non-ordinary isotopes found inVOCs in the exhaled breath, the present invention permits minute amountsof i-AEMs to be used to generate i-EBMs which are readily detectable atthe parts per billion and even at the parts per trillion level inexhaled breath. In addition to the advantage this provides by way ofreducing the mass/volume of AEM required, the use of i-AEMs and thei-SMART device as described herein facilitates monitoring adherenceeither immediately, (Acute Medication Adherence Monitoring, AMAM)several hours (Intermediate Medication Adherence Monitoring, IMAM) oreven several days (Chronic Medication Adherence Monitoring, CMAM) aftera particular medication dose including an i-AEM or i-API is taken or isapplied or administered to a subject. Steady-state concentrations ofAEMs are readily determined (for example using the SMART deviceaccording to this invention and providing careful oversight ofmedication delivery of medication on a regimen designed to reach steadystate levels of AEMs) and related to steady-state EBM concentrations,and, therefore, based on whether a given subject at a given timeexhibits appropriate concentrations of i-EBMs, it can be determinedwhether the subject has taken a particular dose at a particular time,and/or whether over time the subject has been adherent. Intervention cantherefore be undertaken if any departure from the known, calculatedand/or expected pharmacokinetics and pharmacodynamics is detected.

FIGS. 70-74 are instructive with respect to the power of the SMART®system which incorporates the use of a Type II SMART® device accordingto this invention in combination with an i-AEM. Whereas changes inunmarked acetone are barely detectable in the exhaled breath, (as shownin example 26 herein below), the breath kinetics of exhaled d6-acetonefollowing the ingestion of 100 mg of d8-isopropanol per diem for 5 daysis readily followed, as each dose of d8-IPA is reflected in clearlydistinguishable rises in d6-acetone. Deviations from steady state levelsof d6-acetone in the exhaled breath are detectable up to 65 hours afterany given dose of d8-IPA, providing a significant window for confirmingmedication adherence, i.e. IMAM and CMAM.

To further enable and extend IMAM and CMAM, the system according to thisaspect of the invention includes computational features which aredescribed in detail below. The analytical and computational aspects ofthe invention are achieved by the device (Type I, II, III) of thisinvention providing quantitative measurements of EBMs, and, preferablyin real time, comparing pharmacokinetic/pharmacodynamics parametersstored in memory with such EBM measurements. Such computations representa machine implemented software component of the system which, whenintegrated with the given SMART® device and AEM utilized, provides aunitary system for providing definitive medication adherence monitoringover at least dose-to-dose (AMAM) but also over multiple dosages andover multiple days (IMAM and CMAM).

Accordingly, this aspect of the invention provides a method and systemfor using an Adherence Enabling Marker, AEM_(x), (which may be anordinary AEM or an i-AEM), or an Exhaled Drug Ingestion Marker X,EDIM_(x) produced on ingestion or other form of administration orapplication (e.g. topical) of said AEM_(x). The method involvescharacterizing the pharmacokinetics of the particular EDIM_(x) in theexhaled breath of a subject, Y, or in a population of subjects, Z. Thecharacterizing comprises measurement, to within defined confidencelimits utilizing a SMART® detection device (or another device adequateto the task of appropriately defining such parameters for use inconnection with the SMART® device or system as described herein) withsufficient accuracy to provide the parameters described herein below inExample 28.

According to this aspect of the invention, an apparatus for chronicmedication adherence monitoring is provided as a SMART® devicecomprising:

-   A. a sensor selected for accurate detection in the exhaled breath of    at least one subject of at least one Exhaled Drug Ingestion Marker    X, EDIM_(x) produced on ingestion of at least one Adherence Enabling    Marker, AEM_(x);-   B. data storage (as in hard drive, flash drive, EEPROM, in a form    now known or which is developed in the future) operatively coupled    to the sensor, for retention of data generated by the sensor in the    course of characterizing the pharmacokinetics of the EDIM_(x) in the    exhaled breath of a subject, Y, or in a population of subjects, Z;    and-   C. computing means, either in the same unitary device or in a    separate unit to which data obtained as in A and B above is    transmitted or transferred (including, for example, a programmed    central processing unit) which compares each such measurement for    each subject or population of subjects with stored data, as    described herein below, for said subject or population of subjects,    preferably in real time or near real time. For each measurement of    the concentration of EDIMx, a measure of adherence A is generated by    the computing means for each subject.

The characterizing data for storage preferably includes measurementdata, to within defined confidence limits, of:

-   -   a. the Limit of Detection (LoD) of a sensor included in said        device for said marker;    -   b. the background level of said marker or interferents in said        subject or population of subjects;    -   c. the half life of appearance (t_(1/2a)) and elimination        (t_(1/2e)) of said marker from the exhaled breath of said        subject or population of subjects;    -   d. the steady state concentration of said marker in the exhaled        breath at various time points during Adherence Enabling Marker        (AEM) dosing, selected from the group consisting of trough        (C_(Trough,SS)), maximum (C_(MAX,SS)), and other time point post        dosing of the AEM concentrations of said subject or population        of subjects; and    -   e. the time required to attain the maximum concentration        (T_(MAX)) of said marker from the exhaled breath of said subject        or population of subjects.

Such a device according to this invention is preferably configured tointegrate the pharmacokinetic parameters defined above to provide anadherence lookback window, T_(AdhWindow), defined as the period of timerequired for the marker (EDIM) concentration in breath of the subject todecay from an initial value (C_(EDIMo)) to a lower concentration(C_(EDIM,Limit))

$T_{AdhWindow} = {\frac{t_{{1/2}e}}{0.693}*{\ln \left( \frac{C_{EDIMo}}{C_{EDIMLimit}} \right)}}$

wherein:

C_(EDIMo)=original or starting concentration of marker (EDIM) in breathat times equal to or greater than T_(MAX) (i.e., C_(EDIMo)≦C_(MAX)) ofsaid patient;

C_(EDIMLimit)=the final concentration of EDIM in breath of said patient,provided that, if C_(EDIMLimit) denotes the limit of EDIM detection dueto the device LoD or background interference, it would define themaximum T_(AdhWindow); and t_(1/2e)=the elimination half life for saidEDIM.

Such a device preferably exhibits a T_(AdhWindow) between about 1 hourand about 400 hours, and includes a sensor with a LoD for the marker ofbetween 1 part per trillion and 5 parts per billion. In one preferredembodiment, the sensor is adapted to distinguish between ordinary andnon-ordinary isotopes present in EDIMs and volatile compounds whichotherwise would interfere with selective measurement of EDIMs in theexhaled breath. At any time during the T_(AdhWindow) an exhaled breathsample of a subject is obtained and the adherence of the subject to therequired regimen is definitively determined, based on measurement of theconcentration of the EDIM at the time said breath sample or samples areobtained.

9.0 EXAMPLES

Having generally described this invention herein above, the followingexemplary support is provided to further enable those skilled in the artto practice this invention to its full scope. This detailed writtendescription and enabling disclosure is not, however, intended to belimiting on the invention. Rather, for an apprehension of the scope ofthe present invention, those skilled in the art are directed to theappended claims and their equivalents.

Example 1 Hardware Specifications and Performance—Type I Device

General Overview:

The SMART® mGC is capable of detecting aldehydes, ketones, esters,ethers, and miscellaneous volatile organic compounds with, e.g., boilingpoints between 20° C. (68° F.) and 98° C. (208° F.)

FIG. 9 shows a typical output chromatogram detecting key constituents inthe breath, including acetone and isoprene, with clear separation of2-butanone, derived from ingestion of 2-butanol.

In one specific embodiment of the present invention, the SMART® devicehas the following specifications. These specifications are provided toensure a complete and enabling written description of this invention,but those skilled in the art will appreciate that these specificationsshould not be interpreted as limiting on the invention.

-   -   Operating Principle: Isothermal gas chromatography using ambient        air carrier gas and solid-state detector    -   Enclosure Size: 4.1″×8.9″×2.1″ (3.6″ max)    -   Weight: 2.5 lbs. (1.1 kg)    -   Operating Temperature: 10° C. to 34° C.    -   Operating Humidity: 10% to 90% (non-condensing)    -   Storage Temperature: −20° C. to 60° C.    -   Warm-up Time: <10 minutes oven warm-up for analysis    -   Sample: flow activated collection    -   User Interface:    -   Single button push to start    -   Backlit LCD text prompts    -   Audible tone/voice prompts    -   Data Storage: Non-user accessible USB flash drive    -   Color video frame image of user's face.    -   Maintenance: Scrubber replacement on a scheduled basis

Electronic Microcontroller

The SMART® electronic controller resides on a single, multi-layerprinted circuit board and contains, in a preferred embodiment, thefollowing:

-   -   STM 32F107 series 32-bit microcomputer and support circuitry    -   Battery backup for the STM 32F107 clock/calendar    -   16 gigabyte USB memory stick for data storage    -   Voltage regulators and fusing for all circuitry and peripheral        devices    -   Interface circuitry for serial, SPI, and USB communication    -   Pushbutton input    -   Audible sound generation    -   Driver circuitry and connectors for all pumps, valves, fans, and        heaters    -   Analog signal conditioning circuitry for the GC detector,        temperature, pressure, and flow sensors

The controller firmware is written in C or the equivalent and supports ascripting language that allows high-level operating instructions tocontrol the core peripheral and communications drivers, as well assignal processing. The specific sequencing of the SMART GC pumps,valves, heaters, fan, and other peripherals is determined by encrypted,high-level script commands stored on the USB memory stick.

Performance Specifications

The primary performance specifications based upon 2-butanone for theSMART® mGC are:

-   -   Detection Threshold: 5 ppb 2-butanone in breath, (nominal)    -   Measurement Range: 5 ppb to 2.5 ppm of 2-butanone in breath.    -   Carry-Over: <5 ppb 2-butanone equivalent    -   Analyte Retention Time Stability: ±3 to about +5 seconds of        (nominal) retention time (Specific to 2-butanone)

Accessories

-   -   Individually-Packaged Mouthpieces/straws 130.    -   Power Cord    -   Optionally, cellular router, mobile data (e.g., WiFi) hotspot

Patient Population

Patients include those for whom a clinician would like to analyzegaseous samples (e.g., human breath) for suitable organic molecules ofclinical interest (e.g., ingestion of 2-butanol as an AEM).

Environments of Use

The SMART® mGC is intended to be used in a hospital, clinicallaboratory, sub-acute care facility, physician's office, or in the homesetting with or without supervision of a qualified individual.

Materials—Biocompatibility

The following discusses the level and type of patient contact with thedevice and the associated materials.

The SMART® mGC is not in contact, direct or indirect, with the patient,except for the disposable mouthpiece.

The patient only exhales into the mouthpiece of the device, (straw) 130.The straw/mouthpiece 130 in one embodiment is made of ProFax SR 549M, apolypropylene copolymer, or Marlex®, a high-density polyethylene (HDPE).The mouthpiece 130 is commercially available.

Example 2 SMART® mGC Chromatographic Separation of Acetone, Isoprene andEthanol—Type I Device

As shown in FIG. 9, a very clean separation of ethanol, acetone andisoprene is achieved when these compounds are simultaneously adsorbed tothe sample concentrator followed by thermal desorption, separation viathe mGC, and detection by the MOS sensor.

Example 3 Clinical (In Vivo, Human) and Potential Interferenent (InVitro, Benchtop and Clinical) Studies to Optimize and Validate theSMART® System and Composition According to this Invention

To support development and facilitate regulatory filings, a number ofcomplementary in vitro (benchtop: Interference Studies 1 through 4) andclinical (human: Clinical Studies 1 through 4) studies have been carriedout to characterize the SMART® Adherence System. In terms of humanexposure, the system has been safely used to date in 33 human studies(oral, sublingual, and microbicide administration routes), encompassing1,318 experiments in 328 subjects and 8,524 breath analyses. Ofparticular note, three recent prospective, blinded, randomized, crossover clinical validation studies (127 subjects with 472 experiments and2,464 breath analyses) using the SMART® Adherence System designed fororal medications were executed that focused on identifying an optimaladherence-enabling marker (AEM) formulation and carrying out receiveroperating characteristic (ROC) curve analyses to make an optimal cutoffdetermination and assess diagnostic performance (Clinical Studies 1, 2and 3). In addition, a clinical study (Clinical Study 4), examining theimpact of different subject factors on usability, was executed todetermine how patient-friendly the SMART system was in subjects havingdifferent disease states. (e.g., physical, mental, musculoskeletal).

Type I of the SMART® device according to this invention detects a widevariety of volatile organic compounds (VOCs), including but not limitedto alcohols, aldehydes, ketones, esters, and ethers in a qualitative,semi-quantitative, and/or quantitative manner. The ketone, 2-butanone,was selected as a prototypical VOC for detailed device testing accordingto Clinical and Laboratory Standards Institute (CLSI) protocols. Adesktop gas chromatograph (GC), the Hewlett Packard Gas ChromatographModel 5890A, was used as the predicate device. The mGC is operated by atrained individual, and can be used in the health care, clinicallaboratory, or home settings.

The SMART® mGC device is intended to be used by lay people (or, ofcourse, clinications), most frequently in their homes, and willdefinitively document and report, in real-time, adherence to medicationsin the clinical trial or disease management settings. The mGC used inthe SMART® Adherence System was designed to reliably measure e.g.2-butanone in human breath after ingestion of SMART® drugs which have2-butanol, a 2° alcohol that is designated by the FDA as a food additive(generally recognized as safe [GRAS]), incorporated into the dosage formcontaining the active pharmaceutical ingredient (API). The ketone,2-butanone, termed the exhaled drug ingestion marker (EDIM), rapidlyappears in breath after ingestion of the SMART® drug containing2-butanol, due to its efficient enzymatic oxidation by alcoholdehydrogenase (ADH), primarily via the ααADH isoform. The 2-butanol isincorporated into a SMART medication in a manner that has minimal-to-noimpact on the chemistry, manufacturing and controls (CMC) of the API,has no impact on the bioavailability of the API, and does not introduceany extra steps in the clinical trial material (CTM) handling process.The formulation approaches used to incorporate the AEM, 2-butanol, intothe API medication form (e.g., hard gel capsule, powder, or soft gelcontaining 2-butanol) are disclosed herein.

To demonstrate the efficacy and safety of the Type 1 device-based SMART®Adherence Monitoring System, two types of key investigations using hardgelatin study capsules containing 2-butanol were executed:

-   -   1) clinical studies to define:        -   a) optimal configuration of the SMART® System—AEM            formulation in hard gel capsule (Clinical Study 1)        -   b) SMART® System performance (sensitivity, specificity,            accuracy)—AEM formulation in hard gel capsule (Clinical            Study 2)        -   c) optimal configuration of the SMART® System—AEM            formulation in softgel capsule (Clinical Study 3)        -   d) usability of the SMART® System in a simulated home            setting (Clinical Study 4)    -   2) studies to determine the impact of the following potential        interferents:        -   a) new home environment        -   b) ethanol        -   c) cigarette smoking        -   d) various consumer products (e.g., fruit gum, hard candies,            fruit, mouthwash)

These studies and their outcomes are reported here in support of theclaims made with respect to the formulation, and the SMART® AdherenceSystem utilizing the present formulation.

Clinical Studies using hard gelatin study capsules (Clinical Studies 1and 2), and one clinical study using soft gelatin study capsules(Clinical Study 3) were conducted for the SMART® Adherence System.Except where noted in the protocol, all study subjects refrained fromeating, drinking, or smoking for 15 minutes prior to beginning the studyand throughout the duration of the study visit. The timing and type ofrecent food and drink ingestion and cigarette use was documented, alongwith standard subject demographics and past medical history (PMH),medications, and smoking history.

For Clinical Studies 1 and 2, study capsules in sealed opaque Licaps®capsules (AEM formulation placed in sealed size 4 Licaps®, which inturn, was placed within size 0 Licaps®) were made the day of the studyvisit by a certified pharmacy (e.g., Westlab Pharmacy, Gainesville,Fla.) according to the randomization schedule. Licaps® capsules aretwo-piece (cap and body) gelatin capsules that can be specially sealedusing a 50%_(v):50%_(v) ethanol and water mixture to fuse the gelatinedges for secure containment of liquids. Study capsules were used within24 hours of preparation. For the clinical study (Clinical Study 3) usingsoft gelatin study capsules, the soft gelatin 2-butanol formulation wasplaced in an opaque, (e.g., white) size 0 Licaps® capsule. The ethanolformulation was sealed inside an opaque size 4 Licaps® capsule andoverencapsulated in a sealed, opaque size 0 Licaps® capsule (capsule incapsule configuration). The study capsules were used within 5 days ofpackaging by a certified pharmacy (e.g., Westlab Pharmacy, Gainesville,Fla.) according to the randomization schedule.

Each SMART® device had a complete 2-butanone calibration check (0, 10,30, 100, 300, and 1000 ppb 2-butanone standards in breath) at thebeginning and end of the study, whereas a two point 2-butanonecalibration check (0, 10, and 300 ppb 2-butanone standards in breath)was done prior to first use on any given study day unless notedotherwise in a protocol. Calibration data was tracked and recordedthroughout the study. 2-Butanone is detected by the SMART® Device at aretention time of 100 seconds in human breath and causes aconcentration-dependent increase in device response. Data transmissionoccurred using a wireless router.

After each breath into the SMART® Device, a variety of keytime/date-stamped data was stored locally on the device andautomatically uploaded to HIPAA-compliant servers, including but notlimited to: raw signal data, breath chromatogram, yes/no ingestion eventassessment generated from the peak-detection algorithm, photograph ofstudy participant's face for biometric authentication, and SMART® Deviceoperating conditions.

Clinical Study 1 entitled, Clinical Study to Determine the OptimalConfiguration of the SMART GC System, was a prospective, randomized,triple-blind, crossover study in 50 study participants (age 18 years andolder; no known allergies to the study capsule formulations) conductedat the University of Florida. Four (4) hard gelatin formulations werestudied:

-   -   2-butanol (20 mg)    -   2-butanol (20 mg), vanillin (5 mg), DL-menthol (0.7 mg), and        PEG-400 (9.3 mg)    -   2-butanol (40 mg)    -   2-butanol (40 mg), vanillin (10 mg), DL-menthol (1.4 mg), and        PEG-400 (18.6 mg)

The study design consisted of 50 study subjects, each of whom receivedall four formulations (designated as formulations 1, 2, 3, and 4) overfour study visits, each visit consisting of breath sampling intervals atbaseline (0 min: prior to swallowing the capsule) and 10, 20, 30, 45 and60 minutes post-ingestion. Each study subject was randomized to aspecific device for the duration of the study (10 devices; 5 studysubjects per device) and randomized to receive all 4 formulations whichwere self-administered under the supervision of a nurse (directlyobserved ingestion of the study capsule) over 4 study visits with atleast 1 day between visits. This design is consistent with a traditionalpharmacokinetic (PK)-type four period crossover study that assumes nocarryover (i.e., sequence) effect due to adequate separation of thedosing periods (in this case, one day separation).

From the breath chromatograms, measurements of breath concentrations of2-butanone were obtained at baseline (prior to ingestion of the studycapsule) and at 10, 20, 30, 45, and 60 minutes after ingestion of thestudy capsule.

The goal of Clinical Study 1 was to define the optimal operatingconfiguration of the SMART® System using hard gelatin study capsulescontaining 2-butanol. In terms of configuring the SMART® System foroptimal performance, the primary outcome of Study 1 was to determine theoptimal study capsule formulation (dose of 2-butanol and addition ofother ingredients such as flavorants) and the breath kinetics of2-butanone. The outcome measure was 2-butanone concentration (in ppb)recorded repeatedly at each time point during the sampling interval. Thedependent variable for analysis was the change in 2-butanoneconcentration from baseline (Time 0). The change in 2-butanoneconcentration from baseline (“delta over baseline”) provided astatistical adjustment for the potential that some subjects may have arecorded non-zero 2-butanone concentration at Time 0.

Additional analyses (e.g., exploratory covariate analyses in maineffects model) that considered the concomitant effects of demographiccharacteristics (e.g., age, body mass index [BMI], ethnicity, race,gender) and other factors such as the time since last meal wereconducted. Collectively, all of these analyses were considered for thedetermination of the best candidate for study capsule formulation forClinical Study 2.

All analyses and data summaries were performed using SAS Version 9.3.The SAS MIXED procedure was employed for analysis of the two principaloutcome measures. Data was summarized with respect to the following:

-   -   Demographic and other descriptive study subject characteristics        by formulation    -   2-butanone concentrations (“2BC”) by formulation and time    -   Delta over baseline (change from Time 0) 2BC by treatment and        time    -   b) and c) by demographic and other specified factors    -   Extent of 2BC as calculated by the AUC-like “polygon” of values        obtained from the discrete 10 to 60 minute post-ingestion time        points    -   Minimum, mean, and maximum 2BC across time    -   Frequency distribution of time to maximum 2BC    -   Frequency distribution of time to “threshold” 2BC, defined as a        5 ppb, 7.5 ppb, and 10 ppb delta over baseline value

Study participant factors including but not limited to the followingwere analyzed for impact on results (references to Figure nos): 16 aAge; 16 b Gender; 16 c Ethnicity; 16 d Body Mass Index (BMI); 16 e TimeFrom Last Meal; 16 f Alcohol Use; 16 g Tobacco Use; none of thesefactors appeared to be confounding factors (see below).

Results for Clinical Study 1:

2-Butanone Breath Concentration-Time Relationship—Effect ofAdherence-Enabling Marker (AEM) Formulation, see FIG. 17a

Baseline 2-Butanone Concentrations in Human Breath (200 subject visits)

Distribution

-   -   Zero concentrations (<LOD): 190/200 (95%)    -   Non-zero concentrations (>LOD): 10/200 (5%) Central Tendency    -   Mean (SD): 2.4 (19.5) ppb    -   Median: 0 ppb    -   Min, Max: 0, 238.8 ppb

Characteristics of Non-Zero Baseline Concentrations

-   -   One Subject: 3 values (132.4, 238.8, 31.6 ppb)    -   Seven Subjects: 7 values (7.7, 5.1, 5.1, 6.4, 31.3, 17.3, 8.5        ppb)    -   Note: 9/10 and 8/10 of the non-zero values were found in subject        participants with a history of active tobacco and ethanol use,        respectively.

Δ2-Butanone (Change in Concentration from Baseline Values) BreathConcentration-Time Relationship, See FIG. 17 b:

Repeated Measures ANOVA, Main Effect Model

Visit: P=0.12

Formulation: P<0.0001

Time: P<0.0001

AEM Formulation Rank Order: 3>4>1>2

Effect of Adherence-Enabling Marker (AEM) Formulation on Δ2-ButanoneBreath Concentration-Time Relationship: Effect of AEM Formulation;Individual Δ2-Butanone Concentration-Time Curves in 50 Subjects: 20 mg2-Butanol—See FIG. 17c

Ingestion of 40 mg 2-butanol was effective in rapidly generating levelsof 2-butanone in breath that exceeded threshold concentrations. A risein breath 2-butanone levels was detected by the mGC in all 50 subjects.Although significant inter-individual variation in the 2-butanone breathconcentration-time relations was present, 98% and 100% of subjects had2-butanane concentrations 5 ppb threshold at 20 min and 30 min,respectively.

Individual Δ2-Butanone Concentration-Time Curves in 50 Subjects: 20 mg2-Butanol Combo—See FIG. 17 d

Compared to 20 mg 2-butanol, ingestion of the 20 mg 2-butanol comboproduced less favorable 2-butanone concentration-time relations:

-   -   4% (2/50) of subjects (subjects 26 and 33) were        non-responders—no rise in 2-butanone levels was detected in        breath by the mGC over the 60 min study period    -   Greater inter-individual variability was present, including        several cases of 2-butanone exceeding threshold levels only at        later times following ingestion of the formulation        Conclusion: addition of vanillin/DL-menthol/PEG-400 reduced the        prompt appearance of breath 2-butanone, a process likely        attributable to slower 2-butanol release.

Individual Δ2-Butanone Concentration-Time Curves in 50 Subjects: 40 mg2-Butanol—See FIG. 17e

Ingestion of 40 mg 2-butanol was very effective in rapidly generatinglevels of 2-butanone in breath that exceeded all thresholdconcentrations. A rise in breath 2-butanone levels was detected in 100%(50/50) of subjects. Although significant inter-individual variation inthe 2-butanone breath concentration-time relations was present, 98%(49/50) and 100% (50/50) of subjects had 2-butanone concentrationsgreater than all 3 threshold concentrations at 20 min and 30 min,respectively. At 60 min post ingestion, 100% (50/50) of subjects had2-butanone concentrations that persisted above all 3 thresholdconcentrations.

Individual Δ2-Butanone Concentration-Time Curves in 50 Subjects: 40 mg2-Butanol Combo—See FIGS. 17f and 17g

Compared to 40 mg 2-butanol, ingestion of the 40 mg 2-butanol comboproduced slightly less favorable 2-butanone concentration-time relationsbut was still robust in rapidly generating detectable levels of2-butanone in breath:

-   -   2% (1/50) of subjects (subject 18) were non-responders—no rise        in 2-butanone levels was detected in breath by the mGC over the        60 min study period    -   Greater inter-individual variability with more cases of        2-butanone exceeding threshold levels at later times following        ingestion of the formulation; however, 96%, 96%, and 98% of        subjects exceeded the 5 ppb threshold at 20 min, 30 min, and 40        min, respectively, post-ingestion of 40 mg 2-butanol combo.    -   At 60 min post ingestion, 100% (50/50) and 98% (49/50) of        subjects, who ingested formulation 3 (40 mg 2-butanol) and        formulation 4 (40 mg 2-butanol combo), respectively, had        2-butanone concentrations that persisted above the 5 ppb        threshold.    -   Conclusion: addition of vanillin/DL-menthol/PEG-400 slightly        reduced the prompt appearance of breath 2-butanone, a process        likely attributable to slower 2-butanol release, but overall        performance was favorable.

Distribution of 2-Butanone Concentrations by Time, AEM Formulation, andConcentration Threshold Levels; Percent of Subjects (N=50) withΔ2-Butanone Concentrations 5 PPB; See FIG. 17h

Conclusion: Using a 5 ppb rise in 2-butanone levels, 98%/100%/100% and96%/96%/98% of subjects (N=50) exceeded this threshold level at 20, 30and 45 min post-ingestion of 40 mg 2-Butanol and 40 mg 2-Butanol Combo,respectively. Differences among AEM formulations exist.

Percent of Subjects (N=50) with Δ2-Butanone Concentrations 7.5 PPB—SeeFIG. 17i

Conclusion: Using a 7.5 ppb rise in 2-butanone levels, 98%/100%/100% and94%/96%/98% of subjects (N=50) exceeded this threshold level at 20, 30and 45 min post-ingestion of 40 mg 2-Butanol and 40 mg 2-Butanol Combo,respectively. Differences among AEM formulations exist.

Percent of Subjects (N=50) with Δ2-Butanone Concentrations 10 PPB—SeeFIG. 17j

Conclusion: Using a 10 ppb rise in 2-butanone levels, 98%/100%/100% and94%/96%/96% of subjects (N=50) exceeded this threshold level at 20, 30and 45 min post-ingestion of 40 mg 2-Butanol and 40 mg 2-Butanol Combo,respectively. Differences among AEM formulations exist. Δ2-ButanoneBreath Concentration-Time Relationship: Exploratory Analysis ofCovariates

Repeated Measures ANOVA of 2-Butanone Concentration Change from Baseline(T=0 min): Covariates included in the main effects model.

Covariates: Demographics and Timing of Meal

Covariates: age, sex, race, body mass index (BMI), and time since lastmeal

Δ2-Butanone Breath Concentration-Time Relationships: Main Effect Modelwith Factor Covariates - P Values Visit 0.068 Formulation; <0.0001 RankOrder 40 mg 2-butanol > 40 mg 2-butanol combo > 20 mg 2-butanol > 20 mg2-butanol combo Time <0.0001 Age 0.87 Gender 0.45 Ethnicity 0.93 BMI0.91 Time - Last Meal 0.0004

Conclusion: Unlike the time since last meal, demographics had no effecton the appearance of 2-butanone in human breath after the oral ingestionof 2-butanol.

Effect of Meal Timing on D2-Butanone Concentrations Across all AEMFormulations—See FIG. 18a

Conclusion: Eating a meal closer to the time of 2-butanol ingestioncauses a relatively small but significant reduction in 2-butanone breathconcentrations.

Covariates: Tobacco and Alcohol Use—See FIG. 18b

Conclusion: A History of Tobacco (48% Subjects) and alcohol (52%subjects) use by a significant fraction of the study population had noeffect on the appearance of 2-butanone in human breath after the oralingestion of 2-butanol.

ΔT_(MAX): Effect of AEM Formulation—See FIG. 18c

Formulation Key

1, 20 mg 2-butanol (N=50)

2, 20 mg 2-butanol combo (N=48)

3, 40 mg 2-butanol (N=50)

4, 40 mg 2-butanol combo (N=49 See notations * and § in the figure:

*, 2 (subjects 26 and 33) out of 50 (4%) subjects were non-responders(T_(Max)>60 min)

§, 1 (subject 18) out of 50 (2%) subjects were non-responders(T_(Max)>60 min)

Cumulative Frequency (%) of Subjects Achieving ΔT_(Max) by Time andFormulation—See FIG. 18 d.

ΔC_(Max): Effect of AEM Formulation—See FIG. 18e

Formulation Key

1, 20 mg 2-butanol

2, 20 mg 2-butanol combo

3, 40 mg 2-butanol

4, 40 mg 2-butanol combo

Conclusion: AEM formulation had a significant effect on ΔC_(Max).

ΔAUC: Effect of AEM Formulation—See FIG. 18f

Formulation Key

1, 20 mg 2-butanol

2, 20 mg 2-butanol combo

3, 40 mg 2-butanol

4, 40 mg 2-butanol combo

Conclusion: AEM formulation had a significant effect on ΔAUC.

SMART® Device Performance

Study Design: 10 SMART® Devices were assigned to 50 subjects, with 1200total breath analyses=50 subjects×4 visits/subject×*6 breaths/visit.

-   -   Note: 1 breath sample didn't upload (subject 8, visit 2 at 60        min)

5 subjects randomly assigned to use a single device SMART® Deviceperformance

Overall performance: mGC measurements were stable over time and highlylinear in regions (i.e., 2-butanone concentration ranges=0 to 100 and300-3000 ppb) relevant to 2-butanol doses ingested in this study. 2devices were replaced in study; subject 1 after visit 1 (device 212-03):wireless data upload was slow; subject 9 during visit 1: wireless dataupload failed. Devices showed a 1.74× difference in sensitivity to2-butanone in a concentration range=0 to 100 ppb; 7.25% (87 out of 1199breath samples) had 2-Butanone retention times outside the window ofdetection (95 to 105 sec). 1 device (301-06) had camera issues (picturedistortions). The mGC design was modified to address these relativelyminor issues.

SMART ® Device Use Number Time Device Breath % Number of Subjects in Use# Samples Total Assigned to a Device (days) 212-01 114 9.5% 5 (subjects*10, 20, 40, 50) 29 212-03 6 .5% 1 (subject *1) 1 301-01 120 10.0% 5(subjects 2, 12, 22, 32, 42) 29 301-03 120 10.0% 5 (subjects 3, 13, 23,33, 43) 30 301-06 120 10.0% 5 (subjects 4, 14, 24, 41, 44) 29 301-07 12010.0% 5 (subjects 5, 15, 25, 35, 45) 29 301-09 120 10.0% 5 (subjects 9,19, 29, 39, 49) 29 301-10 126 10.5% 6 (subjects 6, *10, 16, 26, 36, 46)29 301-14 96 8.0% 4 (subjects 7, 27, 37, 47) 29 301-16 119 9.9% 5(subjects 8, 18, 28, 38, 48) 29 302-06 138 11.5% 6 (subjects *1. 11, 21,31, 34, 51) 35 TOTAL 1199 100.0%

SMART® Device Performance: Full 2-Butanone Concentration Range—See FIG.18g , which shows the 2-butanone breath concentration-mGC responserelationships by device across the four AEM formulations; relationshipbetween 2-butanone concentration and mGC response is curvilinear (i.e.,square root function), but is highly linear in regions, including lowerconcentrations (0-100 ppb; see next slide) and higher (300-3000 ppb)concentrations relevant to the doses of 2-butanol ingested. Among thedifferent devices, excellent stability over time (<5% variation) withcalibration checks was noted.

Sensitivity of mGC SMART® Devices: Low 2-Butanone Concentrations=0-100Ppb; See FIG. 18h

-   -   Devices were highly linear at low 2-butanone concentrations        (0-100 ppb), which are relevant to yes/no adherence decisions        (rise in concentration=5-10 ppb)    -   Variability in sensitivity present (max slope        ratio=769/443=1.74)—preferably mGC slope is constant across        devices

SMART ® Devices: Retention Time Shifts Retention Time (sec) Out of RangeWithin Range Values Values Number % 95- % Device Breath <95 >105 Total ×105 Total × # Samples sec sec Total Device sec Device 212-01 114 0 0 00.0 114 100.0 212-03 6 2 0 2 33.3 4 66.7 301-01 120 0 0 0 0.0 120 100.0301-03 120 60 0 60 50.0 60 50.0 301-06 120 4 0 4 3.3 116 96.7 301-07 1200 0 0 0.0 120 100.0 301-09 120 23 0 23 19.2 97 80.8 301-10 126 0 0 0 0.0126 100.0 301-14 96 0 0 0 0.0 96 100.0 301-16 119 0 0 0 0.0 119 100.0302-06 138 0 0 0 0.0 138 100.0 TOTAL 1199 89 0 89 7.4 1110 92.6

Of the 1199 breath samples analyzed for 2-butanone content by the SMART®Device, 92.7% (1112 samples) were within the retention time (RT)detection window (95-105 sec). 4 devices accounted for 100% of the 89out of range RT window breath samples, all of which were <95 sec:devices 301-03 (67.4%), 301-09 (25.8%), 301-06 (4.5%), and 212-03(2.2%). A new design version of mGC was created to address this issue.

Study Observations:

AEM Formulations

None of the subjects enrolled in the study reported any significantadverse events, including taste, smell, or gastrointestinal effects.across the four AEM formulations tested.

SMART® Device

-   -   No subject had difficulty providing a breath sample    -   No subject had problems with handling device

Conclusions for Clinical Study 1:

-   -   SMART® Adherence System performance was favorable        -   Biology continues to prove reliable        -   the EDIM (2-butanone) breath PK and its inter-individual            variability across 50 subjects is sufficiently low to permit            the EDIM (2-butanone) to be reliably detected in the breath            of the test population        -   baseline 2-butanone breath concentrations were generally low            in the test population, and did not interfere with reliable            and accurate adherence monitoring utilizing this AEM,            particularly when the baseline breath correction is applied            (i.e., use a rise of breath 2-butanone concentrations of 5            ppb above baseline values)        -   Lead AEM formulation=40 mg 2-butanol combo: 96%, 96%, and            98% detection at a Δ2-Butanone concentration threshold of 5            ppb at 20, 30, and 45 min breath sampling times,            respectively.        -   SMART® devices performed well    -   Appearance of 2-butanone in breath is highly dependent on AEM        formulation:        -   Dose-dependent effect of 2-butanol        -   Combo-dependent effect    -   The SMART system was easily used by subjects and the AEM        formulations were well tolerated    -   Covariate analysis indicates that age, gender, race, BMI, and        chronic alcohol and tobacco use did not affect the generation of        2-butanone in breath after the ingestion of 2-butanol.    -   Based on requirements for SMART® Adherence System performance,        taste masking, long term softgel stability, and softgel        manufacture, the 40 mg 2-butanol combo appears to the “optimal”        candidate AEM formulation.

Clinical Study 2 entitled, Clinical Study to Determine the Sensitivity,Specificity, and Accuracy of the SMART Adherence System, wasprospective, randomized, triple-blind, placebo-controlled, cross-overstudy in 30 volunteers (age 18 years and older; no known allergies tothe study capsule formulations) conducted at the University of Florida.

The study was designed to determine the sensitivity, specificity, andaccuracy of the SMART® System using hard gelatin study capsulescontaining 2-butanol. The primary study objective was to determine thediagnostic accuracy of the SMART® Breath Monitoring System indistinguishing between the ingestion of study capsules containing2-butanol versus placebo capsules containing the same amount of ethanolinstead of 2-butanol. The associated ingredients, (i.e., vanillin,DL-menthol, and PEG-400), were the same for both study capsuleformulations.

A single formulation of the study capsule, namely Formulation 4 (i.e.,2-butanol [40 mg], vanillin [10 mg], DL-menthol [1.4 mg], and PEG-400[18.6 mg]) was studied; each study subject was randomly assigned toingest two types of capsules, namely a capsule containing 2-butanol[SMART capsule], and a capsule containing the same mass of ethanol andassociated excipients as that used for the SMART capsule (placebocapsule).

Each study subject was randomized to receive a total of 3 SMART®capsules and 3 placebo capsules (50%:50% randomization with the capsuletypes) under the supervision of a nurse (directly observed ingestion)over 6 study visits with at least 1 day between visits. Thus, ClinicalStudy 2 contained a total of 180 study visits.

Each study subject was randomized to one 1 of 30 SMART® Devices for theduration of the study. Breath samples were obtained at baseline(pre-ingestion) and at 10, 20, and 30 minutes after ingesting the studycapsule.

Device calibration data was tracked and recorded by Expert 1. Expert 2manually read data outputs in a blinded manner and determined whether aSMART® or placebo capsule was ingested. At study completion, theassessments made by Expert 2 was compared against those automaticallymade by the SMART® Device using the optimized configuration derived fromStudy 1 (e.g., optimized formulation, time of breath sampling, proposeddelta 2-butanone concentration cut-off levels).

The sample size, the optimal formulation, and timing of breath samplingused in Clinical Study 2 were determined based on the analysis ofClinical Study 1 results. Since the post-baseline 2-butanone breathconcentration levels in study subjects who ingested the placebo capsulewere observed to be close to zero (below the limit of detection) and thebreath concentrations in study subjects who ingested the hard gelatinstudy capsule containing 2-butanol were well above 5 ppb, the differencein proportions of study subjects above this and even higher thresholdsbetween the 2-butanol study capsule and placebo study capsule was quitelarge. This study enrolled 30 completed subjects to provide a soundframework for the estimation of normal distribution-based statistics.

Statistical analysis of the data from Clinical Study 2 was handled in amanner similar to that described in Clinical Study 1. The dependentvariable was the change in 2-butanone breath concentration from baselinevalues. Performance metrics of the SMART® System were based onsensitivity/specificity analysis and accuracy determination endpoints.Analysis followed the guidance from the Clinical and LaboratoryStandards Institute (CLSI) EP24-A2, entitled “Assessment of theDiagnostic Accuracy of Laboratory Tests Using Receiver OperatingCharacteristic (ROC) Curves”. To assess the effectiveness of the SMART®System, ROC curves (plots of Se versus 1-Sp) were used to summarize thediagnostic performance of the SMART® System at 10, 20, and 30 minutesafter study capsule ingestion using an automated detection algorithm(software) and an expert manual reader. Since the distributional natureof the 2-butanone breath concentration data is such that results aremore dichotomous in nature (e.g., virtually close to zero orsufficiently greater than 10 ppb), sensitivity/specificity analysis used2×2 tables where the relative sensitivity/specificity of the SMART®System was assessed at various concentration thresholds.

Data was summarized with respect to the following:

-   -   Demographic and other descriptive study participant        characteristics by formulation    -   2-butanone concentrations (“2BC”) by formulation and time    -   Delta over baseline (change from Time 0) 2BC by treatment and        time    -   b) and c) by demographic and other specified factors    -   Extent of 2BC as calculated by the AUC-like “polygon” of values        obtained from the discrete 10 to 30 minute post-ingestion time        points    -   Minimum, mean, and maximum 2BC across time    -   Frequency distribution of time to maximum 2BC (over the 30 min        time frame)    -   Frequency distribution of time to “threshold” 2BC, defined as a        5 ppb, 7.5 ppb, and 10 ppb delta over baseline value

Results for Clinical Study 2:

A total of 33 subjects (3 did not complete all visits for reasonsunrelated to the study) participated in Clinical Study 2. Thirty three(33) subjects received placebo capsules, whereas 31 received SMART(2-butanol AEM formulation) capsules. A total of 184 visits (93 placebo,91 SMART) were included in the analysis.

Summary statistics for the demographics are shown in the Table below:

SMART Placebo Variable (2-Butanol) (Ethanol) Gender [N (%)] Male 16(51.6) 18 (54.5) Female 15 (48.4) 15 (45.5) Ethnicity [N (%)] White 22(71.0) 24 (72.7) Black (African  7 (22.6)  7 (21.2) American)Asian/Other 2 (6.5) 2 (6.1) Age (yrs) N 31 33 Mean(SD) 47.8 (13)    48.4(12.9)  Median 52 52 Min, Max 25, 64 25, 64 BMI (kg/m²) N 31 33 Mean(SD)28.1 (6.2)   28.4 (6.6)   Median   26.6   26.6 Min, Max 19.8, 41.6 19.8,42.6

Using a Δ2-butanone concentration cutoff value of 5 ppb, 181/184 (98.4%)intent to treat (ITT) cases were interpreted correctly by the SMART®Adherence System. Of the 3 cases not interpreted correctly, there was 1false positive and 2 false negatives.

Breath Sampling Time 10 min 20 min 30 min Overall Accuracy 82.6% 94.6%98.4% 98.4% Sensitivity 64.8% 90.1% 96.7% 97.8% Specificity  100% 98.9% 100% 98.9%

As shown in this table, the optimal breath sampling time after ingestingthe capsule containing the AEM formulation (2-butanol) was 20 to 30 minwhere accuracies were approximately 95% and higher.

Conclusions for Clinical Study 2:

The SMART® Adherence System is highly accurate.

Clinical Study 3, designed on the basis of the results from ClinicalStudies 1 and 2, is entitled, Clinical Study to Determine the OptimalConfiguration of the SMART® Breath Monitoring System Using Soft GelatinSMART® Capsules (see also Clinical Study 4 below), was conducted todetermine the optimal configuration of the SMART® Breath MonitoringSystem using soft gelatin study capsules containing 2-butanol. The goalsof this study were: 1) to establish the optimal cutoff 2-butanone breathconcentration (e.g., increase of 5 ppb above baseline values) usingReceiver Operating Characteristics (ROC) curves analysis, 2) todetermine the SMART® Breath Monitoring System sensitivity, specificity,and accuracy at the optimal 2-butanone cutoff breath concentration, 3)to determine the range of optimal breath sampling time(s) (i.e., 20, 30,40, 60, and 90 minutes) following 2-butanol study capsule ingestion, and4) to establish the duration of 2-butanone persistence in breath.

A single formulation of the soft gelatin study capsule, (i.e., 2-butanol[40 mg], vanillin [10 mg], DL-menthol [1.4 mg], and PEG-400 [18.6 mg])was studied. Each subject was randomly assigned to ingest two types ofcapsule formulations over 2 subject visits: 1) a capsule containing2-butanol (SMART® Capsule); and 2) a placebo capsule containing ethanol.The placebo capsule contained the same mass of ethanol and associatedexcipients as used in the 2-butanol capsule. Ingestion of a capsule ateach subject visit was verified through direct observation (i.e.,directly observed therapy [DOT]) by the Clinical ResearchCoordinator(s).

44 subjects were enrolled and a total of 88 adherence assessments (44capsules containing 2-butanol and 44 placebo capsules) were made usingthe SMART® Breath Monitoring System.

44 mGC units were employed in the study. A given subject was randomlyassigned a specific mGC for use during both study visits. After abaseline breath sample was obtained (t=0 minutes), the subject ingestedone study capsule (SMART or placebo), and then provided breath samplesat 20, 30, 40, 60, and 90 minutes after ingestion of the capsule.

The sample size, the optimal formulation, and the timing of breathsampling were determined based on the analysis of Clinical Study 1results of hard gelatin SMART® Capsules.

The outcome measure was 2-butanone concentration (in ppb) recordedrepeatedly at each time point during the sampling interval. Thedependent variable was the change in 2-butanone breath concentrationfrom baseline (Time 0) values. The change in 2-butanone concentrationfrom baseline (“delta over baseline”) provided a statistical adjustmentfor the potential that some subjects may have a recorded non-zero2-butanone concentration at Time 0.

Performance metrics of the SMART® Breath Monitoring System were based onReceiver Operating Characteristic (ROC) curves analysis, including2-butanone cutoff determination (e.g., 5 ppb rise above baselinevalues), sensitivity/specificity analysis, and accuracy determinationendpoints. Analysis followed the guidance from the Clinical andLaboratory Standards Institute (CLSI) EP24-A2, entitled “Assessment ofthe Diagnostic Accuracy of Laboratory Tests Using Receiver OperatingCharacteristic Curves”. To assess the effectiveness of the SMART® BreathMonitoring System, ROC curves (plots of Se versus 1-Sp; and plots ofcutoff concentrations versus Se and Sp) were used to summarize thediagnostic performance of the SMART® System at 20, 30, 40, 60, and 90minutes after capsule ingestion at a single cutoff 2-butanone breathconcentration (e.g., 5 ppb rise above baseline values), using anautomated detection algorithm (software) and the manual mGC reader.

Data was summarized with respect to the following:

-   -   Demographic and other descriptive study subject characteristics    -   2-butanone concentrations (“2BC”) by formulation (placebo and        SMART® Capsules)    -   Delta over baseline (change from Time 0) 2BC by formulation        (placebo and SMART® Capsules)    -   b) and c) by demographic and other specified factors    -   Extent of 2BC as calculated by the AUC-like “polygon” of values        obtained from the discrete 20 to 90 minute post-ingestion time        points    -   Minimum, mean, and maximum 2BC across time    -   Frequency distribution of time to maximum 2BC    -   Frequency distribution of time to “threshold” 2BC, defined as        the single BC cutoff concentration (e.g., 5 ppb delta over        baseline values)

Results for Clinical Study 3:

A total of 44 subjects participated in Clinical Study 3. All subjectscompleted both visits, and therefore randomly received a placebo capsuleand SMART (2-butanol AEM formulation) capsule over two visits. Thus, atotal of 88 visits were included in the analysis. Summary statistics forthe demographics are shown in the below table:

Characteristic Parameter DR 0054 Age (Years) n 44 Mean 40.34 Std. Dev.18.16 Median 39.00 Minimum 19.0 Maximum 75.0 BMI (kg/m2) n 44 Mean 25.75Std. Dev. 4.93 Median 24.95 Minimum 17.4 Maximum 39.5 Time - Meal (hrs)n 44 Mean 6.37 Std. Dev. 5.98 Median 3.00 Minimum 0.5 Maximum 19.5Gender n (%) Male 20 (45.45) Female 24 (54.55) Tobacco Use n(%) Yes 16(36.36) No 28 (63.64) Alcohol Use n(%) Yes 33 (75.00) No 11 (25.00)

With a Δ2-butanone concentration cutoff concentration of 5 ppb, theresults of SMART® performance using the soft gel-based capsulecontaining the AEM (2-butanol) formulation are depicted in the Tablebelow. Although the SMART® Adherence System continues to be highlyaccurate, the soft gel capsule-based delivery of the AEM (2-butanol)appears to be slower in releasing the 2-butanol in the stomach relativeto the hard gel capsule-based approaches used in Clinical Study 1 and 2.In the latter case (hard gel capsule), a breath sampling time of 20 to30 min was associated with high accuracy, whereas in the former case(softgel capsule), breath sampling times of 40 min and longer arerequired.

Breath Sampling Time (N = 44 subjects) 20 min 30 (min) 40 (min) 60 (min)90 (min) Accuracy 76.1 87.5 92.0 97.7 94.3 Sensitivity 52.3 75.0 84.095.5 97.7 Specificity 100 100 100 100 90.9

With regard to adverse events, across the 88 visits with 44 subjects,only 4 reports of taste and/or mild “stomach tingling or upset” werenoted in four different subjects (subjects 0054-01, 0054,03, 0054-07,and 0054-30)—all of which received placebo (ethanol containing)capsules. In other words, no adverse events, including reports of mildtastes/smells and/or gastrointestinal issues, were reported in anysubjects ingesting softgels containing the AEM (2-butanol) formulation.

Conclusions for Clinical Study 3:

The SMART Adherence System using softgels to deliver the AEM (2-butanol)is highly accurate, but requires longer breath sampling times to do so.

Clinical Study 4 entitled, Usability Validation Study of the SMART®Adherence Device, fulfilled the validation plan activity identified inSection 5.6 of the IEC 62366:2007 International Standard, Application ofUsability Engineering to Medical Devices. Additional guidance wasobtained from the draft document Guidance for Industry and Food and DrugAdministration Staff: Applying Human Factors and Usability Engineeringto Optimize Medical Device Design.

Twenty-five (25) study subjects who represent potential users of theSMART® Device were enrolled in this study, conducted at a typical marketresearch facility (Jackson Associates Research Facility, Woburn, Mass.)which was mocked up to represent a typical home environment in whichusers would interact with the device.

The purpose of this study was to validate the usability of the SMART®Device and its accompanying user documentation. The study objectiveswere to: 1) demonstrate that the SMART® Device can be set up and used byrepresentative users under simulated use conditions without producingpatterns of failures that could result in a negative impact or injury tothemselves, 2) verify that the device documentation and trainingprovided as part of this study are effective, 3). ensure that thepotential use-related safety issues associated with using the devicewere adequately mitigated, and 4) verify whether the validation successcriteria were met.

Study subjects received the expected training that users would receiveprior to use. Test sessions occurred no sooner than one day after thetraining session for all individuals. Study subjects were presented atask scenario and asked to work through various subtasks with the deviceindependently.

The test moderator recorded completion rates and noted positive andnegative comments, usability issues, errors, and number of timessubjects required assistance to use the device appropriately. Followingthe completion of all tasks, the moderator conducted a separate,in-depth interview to gather more detailed understanding of any observeduse errors, usability problems, and near misses.

The study results reported the successes and the extent of failures forall listed tasks. Each instance of task failure was evaluated todetermine its root cause. Every study subject who experienced adifficulty or a failure was interviewed about that difficulty or failureto determine the cause from the study subject's perspective. Directperformance data were used for support. Observation notes, videorecordings, and procedure artifacts were used, if necessary. Dataanalysis also included subjective feedback regarding critical taskexperience, difficulties, “close calls,” and any task failures by studysubjects.

Both performance-based and subjective data were further analyzed toensure that no new risks were identified. A determination was made ifany of the investigated failures would have led to user harm. Followingthe test, the objective and subjective measures were analyzed and anyusability or safety issues with the User Guide or Quick Reference Cardwere identified.

Xhale Smart analyzed any failures uncovered in this testing and updatedthe risk analysis. The follow-up risk analysis used the same approachthat Xhale Smart took in the course of its prior risk managementassessments, leading to the final disposition of use errors andusability issues as acceptable or not. The failures were described, aswell as whether or not failures that occurred were associated with thedesign of the device, its labeling or documentation system and theextent of the association. The analysis of residual risk determined ifdesign modifications were indicated or if not, the analysis demonstratedthe impossibility or impracticality of reducing these risks further andthat the residual risk was outweighed by the benefits offered by thedevice. If design modifications were indicated, and were significant,they were implemented and validated.

Results and Conclusions for Clinical Study 4:

In terms of usability, the SMART® Device was validated

1) Test tasks were successfully completed according to the successcriteria, and all failures were investigated and shown to not have ledto subsequent user or healthcare professional harm, and

2) No safety-related errors or usability issues were noted that could befurther mitigated through design, training, or labeling, and none of theobserved use errors, near misses, and usability issues (if any wereobserved) presented an unacceptable risk to the safety and effective useof the device.

Summary of Clinical Validation Work (Clinical Study 1, 2, 3, and 4)

In summary, the Type 1-based SMART system was found to be not onlypatient friendly in terms of usability across a wide range of diseasestates, but its performance was also favorable across a wide range ofsubject factors, including age, gender, race, body mass index (BMI),disease conditions, and time of food ingestion, and even in populationsenriched with subjects who chronically consumed alcohol and/or usedtobacco products.

Specifically, after ingestion of the gelatin capsules containing anoptimized AEM formulation, the following notable clinical findings werefound: 1) greater than 98% of subjects gave an overall positive response(detection of breath marker by the Type 1 SMART® Device), and 2)adherence accuracies exceeding 95% can be achieved when a 20-90 minbreath marker detection window is employed. Given the above results, weconclude that the SMART® Adherence System holds significant promise as anovel technology to definitively measure and monitor medicationadherence in various clinical settings.

Example 4 Interference Studies to the SMART Adherence System Using2-Butanol as the AEM and a Type 1 Device

A series of four experiments were carried out to evaluate the effect ofpotential interferents on the function of the SMART® Adherence Systemutilizing a Type I Device and 2-butanol as the AEM. For the sake ofbrevity, summaries of results and conclusions are provided. As shownbelow and illustrated above in Clinical Study 1, 2, and 3 (excellentaccuracy across diverse subject populations enriched with smokers,ethanol drinkers, and enrollees fed ad lib), the Type I device-basedSMART® system can perform well, even in the presence of a wide varietyof consumer products, ethanol, cigarette smoking.

These four interferents were selected for study, because they weredeemed to have the highest likelihood of reducing the efficacy of a TypeI device-based SMART® Adherence System using 2-butanol as the AEM. Wepreviously reported that food (e.g., yogurt, cheddar cheese, black tea,tomatoes), which contains the greatest content of 2-butanol and/or2-butanone, and the fed state did not affect the SMART® Adherence System(Morey et al, Oral Adherence Monitoring Using a Breath Test toSupplement Highly Active Antiretroviral Therapy, AIDS Behav17(1):298-306. 2013).

Interference Study 1: New Home Environment

SUMMARY

The purpose of this study was to evaluate indoor air samples from fiverecently built homes for the presence of volatile organic compounds(VOCs) that could potentially interfere with the function of the2-butanone-based SMART® mini-gas chromatographs (mGC) System byproviding an additional source of VOC with a retention time (100±5 sec)similar to that of 2-butanone on the mGC. Indoor air from each home wastested on-site using four separate SMART® mGCs. Paired air samples werecollected from each home to confirm the identity of the VOCs present,using tandem gas chromatography mass spectrometry (GC/MS). All homescontained VOCs that are typically associated with the use ofconstruction materials (e.g., acetone, isopropyl alcohol, butanal, and2-butanone). The 2-butanone levels measured by the SMART® mGCs (identityverified by GC/MS) in the ambient indoor air from the new homes was low(range: 5.9 to 16.5 ppb) and could potentially contribute to themeasured breath 2-butanone concentrations of home residents. However, atleast three reasons exist that substantially mitigate the potential ofthe new construction environment to adversely impact the function of theSMART® mGC system: 1) the SMART® mGC system uses a baseline breathsample to measure background concentrations of 2-butanone concentrationsin breath, 2) the equilibration between ambient concentrations of2-butanone levels in the air and those in the blood of humans occurrelatively slowly (e.g., hours),^(4,5) and 3) much higher levels of2-butanone are typically generated in breath, relative to those found inhomes with new construction, following ingestion of AEM, 2-butanol.

INTRODUCTION

The SMART® miniature gas chromatograph (mGC) measures the concentrationof 2-butanone in exhaled breath following the ingestion of the AEM,2-butanol. Therefore, specific volatile organic compounds (VOCs),previously identified in a previous study (internal Xhale DocumentDR-0026), which have retention times on the SMART mGC similar to2-butanone (i.e., 100±5 seconds) have the potential to interfere withthe performance of the SMART® mGC Adherence System.

Materials used in home construction including paints, sealants,synthetic or laminated flooring, carpeting and other furnishings, mayrelease VOCs in the home environment. The highest levels (i.e., worstcase scenario) of VOCs in indoor home environments are found during themonths immediately following the home construction.^(1,2,3) Thosematerials that release 2-butanone or other VOCs with retention timessimilar to 2-butanone on the SMART® mGC (i.e., 100±5 seconds from thatof 2-butanone), could introduce interfering VOCs into the breath of newhome residents, and alter the real (2-butanone) or apparent(non-2-butanone VOC with a retention time between 95 to 105 sec)mGC-derived concentration of 2-butanone in breath. The purpose of thisstudy was to sample indoor air from new homes (i.e., worst casescenario) on site using the SMART® mGC and GC/MS and to screen for thepresence of VOCs that could potentially interfere with the2-butanone-based mGC SMART® Adherence System.

Materials and Methods

Test Articles and Formulations

Four SMART® mGCs were used for this study to detect of 2-butanone indoorair of new homes. The SMART® mGCs with serial numbers 100113060024,100113060028, 100113060033, and 100113060047 were used in the study.These units are identified as 600-24, 600-28, 600-33, and 600-47,respectively, in this report.

1-L Tedlar gas sampling bags used for standard preparation werepurchased from SKC Inc. (Eighty Four, Pa.). A single 10.0 mL Hamilton(Reno, Nev.) gas-tight syringe (Model Number 1010) was used for thedilution of the 2-butanone gas standard.

GC/MS samples were collected on stainless steel Tenax TA sample tubes(Model # C1-AXXX-5003) manufactured by Markes International Incorporated(Cincinnati, Ohio) using a 100 mL Hamilton (Reno, Nev.) gas-tightsyringe (Model #1100).

Reference standards for the four SMART® mGCs and the Thermo ISQ GC/MSwere completed at the University of Florida Innovation Hub on Sep. 24,2013. A National Institute of Standards and Technology (NIST) certified2-butanone gas standard was diluted into Tedlar gas sampling bagscontaining dry ultra-high purity (UHP) nitrogen to create standardscontaining 2-butanone concentrations of 0, 10, 30, 100, 300, and 1000ppb. The 2-butanone calibration curves generated from these standards oneach of the four SMART® mGCs and the GC/MS were created.

Study Design

Indoor air samples from five homes (identified in this report as Homes1-5) were analyzed using the four SMART® mGCs. A single room air sampleof ˜30 mL was taken and automatically analyzed by the individual mGCs ateach location. One paired sample was collected from each of the fivehomes (Homes 1 through 5) for tandem gas chromatography massspectrometry (GC/MS), in order to qualitatively assess and identify theVOCs in the indoor environment. Air samples collected for the GC/MSanalysis were prepared by drawing 50 cc of air through a clean Tenax gassampling tube over 1 minute using a 100 mL gas tight syringe.Immediately after taking the sample, the tube was capped and theidentification number of the tube recorded. The tubes were returned tothe Innovation Hub for GC/MS analysis.

The homes used for this study were new-constructions (never occupied),and contained similar materials and fixtures (e.g., painted, flooring,cabinets) that had been installed within 30 days of sample collection.Homes 1 and 2 were located in the same housing development and weremanufactured by the same builder. A different developer manufacturedHomes 3, 4 and 5. Homes 3 and 4 were located in same neighborhood, andHome 5 was located in a separate development. No information wascollected to identify the specific materials used in construction.

Data Storage and Processing

All SMART® mGC data was automatically collected on the device andsubsequently transmitted to and stored on the Xhale Inc. securedservers. First derivative plots for the standards and breath samplescollected by the SMART® mGCs were imported into Microsoft Excel® todetermine 2-butanone peak height and retention times. GC/MS data wascollected on the instrument's control computer and are stored on compactdisk. GC/MS chromatograms were analyzed using Thermo ScientificXcaliber® software. VOCs were identified by matching collected massspectra to corresponding library spectra in the NIST database.

Statistical Analyses

For the interference screen, potential interferents were defined asthose VOCs that generate a measured 2-butanone response of ≧5 ppb on theSMART® mGC. Data are expressed as mean±standard deviation. The effect ofnew home environments on the 2-butanone concentrations measured in airby the SMART® mGC were evaluated using a two-way analysis of variance(ANOVA) (factors: home and device) (SigmaPlot 11.2, Systat Software,Inc., San Jose, Calif.). P values <0.05 were considered statisticallysignificant.

Results and Discussion

All SMART® mGCs used in this study responded similarly to theenvironmental VOCs in each home tested. The mean 2-butanone levelsmeasured in the indoor air from the five homes ranged from 5.9-16.5 ppb.These levels are consistent with the indoor air 2-butanoneconcentrations measured in new site-built homes by Lindstrom et al (1-33ppb)³ and Hodgson et al (2.4-42.1 ppb with an average concentration of8.8 ppb).² The 2-butanone measurement was similar among the fourdifferent devices (p=0.71) and the coefficient of variation (CV) inmeasured 2-butanone concentration across the four SMART® mGCs was <15%at each location.

A qualitative analysis of the environmental air by GC/MS, was used toidentify VOCs present in the indoor air of each home. Representativechromatograms (e.g., from Home 2) were created, and the identities ofthe VOCs observed on the SMART® mGC and the GC/MS confirmed. 2-butanonewas identified by GC/MS in all the homes tested (i.e., 5/5). The VOCsidentified by the GC/MS in the homes sampled in this study, areconsistent with the low levels of VOCs commonly found in constructionmaterials: 1,3-dimethylcyclohexane, 1-butanol, 1-methoxy-2-propanol,1-pentanol, 1-pentene, 2,2-dimethylhexane, 2-butanone,2-methyl-1-propanol, 2-methylheptane, 2-methylhexane, 2-methylpentane,2-propoxyethanol, 3-methyheptane, 3-methylhexane, acetone,acrylonitrile, benzene, butanal, butyric acid, chloroform, cyclohexane,cyclopentane, ethanol, ethyl acetate, hexanal, hexane, isobutyl alcohol,isobutyl ether, isoprene, isopropanol, methyl vinyl ketone,methylcyclohexane, methylcyclopentane, methyisopropylketone, n-butylacetate, n-propyl acetate, pentanal, pentane, pentyl alcohol, propanoicacid, propylene glycol, tetrahydrofuran, and toluene.

There are two processes by which VOCs from ambient air could appear inexhaled breath. The first is by being exhaled from the lungs immediatelyafter inhalation (i.e., the VOC does not partition out of the inhaledair and remains in the vapor phase during exhalation). The second isthrough absorption of the VOC from the lungs into blood and body tissuesfollowed by a later partitioning from blood or tissues back into exhaledbreath. Uptake kinetics of 2-butanone for inhalation exposure have beenstudied in human subjects exposed to high concentrations of 2-butanone(100,000-200,000 ppb)^(4,5) and indicate that, at least for 2-butanone,both of these processes occur simultaneously to varying degrees and takehours to attain equilibrium. These studies reported pulmonary uptake of2-butanone from air of 70%, and exhaled air 2-butanone concentrationsranged between 6%⁵ and 50%⁴ of the inhaled concentration. It isimportant to note that regardless of the extent of uptake, ambient VOClevels represent the highest concentration that will appear in exhaledbreath from inhalation exposure alone.

CONCLUSIONS

The 2-butanone levels measured by the SMART® mGCs in the indoor air offive new construction homes ranged between 5.9 and 16.5 ppb. Given thesensitivity of the SMART® mGC, the indoor air 2-butanone leveldetermined in this study may contribute to the measured breath2-butanone concentrations of home residents. However, the risk of thenew home environment causing inaccurate (i.e., false positive or falsenegative) results readying by the SMART® Adherence System is minimal forat least 3 reasons. First, the SMART® mGC system is capable of using abaseline breath sample to measure background concentrations of2-butanone concentrations in breath, Second, the equilibration betweenambient concentrations of 2-butanone levels in the air and those in theblood of humans occur relatively slowly (e.g., hours),^(4,5)

Interfering VOCs from ambient air in new homes should contribute equallyto the baseline and sample breaths. Since the SMART® System monitors thechange in exhaled 2-butanone by subtracting the baseline levels (priorto ingestion of the AEM from the sample breath, constant backgroundlevels of 2-butanone should be effectively eliminated from thedetermination. Third, much higher levels of 2-butanone are typicallygenerated in breath, relative to those found in homes with newconstruction, following ingestion of the AEM, 2-butanol.

REFERENCES

-   [1] Retrieved from http://www.epa.gov/iaq/voc.html-   [2] Hodgson, A. T., Rudd, A. F., Beal, D., Chandra, S., Volatile    Organic Compound Concentrations and Emission Rates in New    Manufactured and Site-Built Houses, Indoor Air, (2000) 10(3):    178-92.-   [3] Lindstrom, A. B., Proffitt, Effects of modified residential    construction on indoor air quality, Indoor Air, (1995) 5, 258-269-   [4] Liira, J., Riihimaki, V., Engstrom, K, Pfaffli, P., Coexposure    of man to m-xylene and methyl ethyl ketone—Kinetics and metabolism,    Scand J Work Environ Health, (1988) 14(5):322-327.-   [5] Dick, R B; Brown, W D; Setzer, J V, Effects of short duration    exposures to acetone and methyl ethyl ketone, Toxicol Lett, (1988)    43:31-49.

Interference Study 2: Cigarette Smoke

SUMMARY

Cigarette smoke is known to contain a large number of volatile organiccompounds (VOCs), many present at high concentrations. Compoundsintroduced in human breath as a result of smoking events (e.g.,2-butanone, ethyl acetate) could potentially interfere with the functionof the 2-butanone-based SMART® mini-gas chromatographs (mGC) System byproviding an additional source of VOC with a retention time (100±5 sec)similar to that of 2-butanone on the mGC. The objectives of this studywere twofold: 1) evaluate the presence of potential interferents in thehome environments (n=5 homes) of smokers, and 2) screen breath samplesfrom smokers (n=5 volunteers) for potential interferents from smokingtwo commonly used cigarette brands (i.e., Newport and Marlboro). Thekinetics of potentially interfering breath VOCs from five (5) studysubjects were evaluated following smoking events (T=0, 10 and 15minutes), in support of a plan to understand and mitigate potentialrisks of smoking causing detrimental effects on SMART® mGC Systemperformance.

Screening of VOCs from the home environments showed that only one of thefive (1/5) homes had a mean indoor air 2-butanone concentration 5 ppb(mean concentration 6.9 ppb). Smoking did not result in a clinicallysignificant change (i.e. 5 ppb) from the baseline breath 2-butanoneconcentration on the SMART mGC, in any of the study participants. Anypotential risk of inaccurate 2-butanone results in human breath fromsmoking can be adequately mitigated by 1) collecting a baseline breathsample prior to ingestion of the AEM, 2-butanol, and 2) having a 15minute wait period from smoking, before a subject breath sample isgiven. This finding is consistent with the results of clinical studies(Example 3, Clinical Study 1, 2, and 3) investigating the performance ofthe SMART® mGC System, which demonstrated favorable performance, even insubject populations enriched with participants having a significantsmoking history.

INTRODUCTION

The Centers for Disease Control and Prevention estimates thatapproximately 19% of the adult American population smoke cigarettes.¹Cigarette smoke contains over 4400 compounds including 2-butanone.^(2,3)Volatile organic compounds (VOCs) present in cigarette smoke (i.e.,2-butanone, ethyl acetate) may interfere with the SMART® mGC System byhaving similar retention times (i.e., 100±5 seconds) as the breathmarker, 2-butanone, that is generated after ingestion of the AEM,2-butanol. The purpose of this study was to screen VOCs associated withsmoking two widely used cigarette brands (i.e., Newport and Marlboro)that could potentially interfere with SMART® mGC function. Specifically,these cigarettes may release 2-butanone or other VOCs with retentiontimes similar to 2-butanone on the SMART® mGC, into the breath ofsmokers (and passive non-smokers), and alter the real (2-butanone) orapparent (non-2-butanone VOC with a retention time=95 to 105 sec)mGC-derived concentration of 2-butanone in breath. The kinetics(time-dependent behavior) of these potential interferents in humanbreath were evaluated in support of a plan to understand and mitigatepotential risks of smoking causing detrimental effects on SMART® mGCSystem performance.

Materials and Methods

Test Articles and Formulations

Five Xhale mGCs were used for this study. One mGC device was used perperson, and the instruments were randomly assigned to each individual.The mGCs used had serial numbers 100112120003, 100113030039,100113030041, 100113030043, and 100113030044. These units will beidentified as 212-03, 303-39, 303-41, 303-43, and 303-44 in this report,respectively.

1-L Tedlar gas sampling bags were purchased from SKC Inc. (Eighty Four,Pa.). Each bag was used only once. A single 10.0 mL Hamilton gas-tightsyringe (Model Number 1010, Fisher Scientific part number 14-815-183)was used for the dilution of the 2-butanone gas standard.

2-Butanone standards were created by diluting appropriate aliquots of aprimary NIST certified dry nitrogen 10 ppm 2-butanone gas standard(Matheson Tri-Gas MICRO MAT 58 Item Number GMT2677977TH, Lot Number109-26-07599, Expiration Date 5/11/14) into 1-L Tedlar bags containingblank breath. The two cigarette brands used for this study (Marlboro andNewport) were purchased from a Publix Supermarket in Gainesville Fla. onMay 3, 2013. These brands were chosen based on cigarette brandpreferences reported for the general smoking population and representthe two most popular brands of cigarettes in the United States.4

Butanone Standard Creation and Analysis

Dilution of a NIST-certified 2-butanone gas standard into Tedlar gassampling bags containing a blank breath sample was performed to create astandard curve at four concentrations (0, 10, 100, and 1000 ppb). Thestandard curve for 2-butanone was analyzed on each of the four SMART®mGCs used in this study, at the Nanoscale Research Facility of theUniversity of Florida.

Investigational Plan

All samples (i.e., indoor and breath) for this study were collected inthe individual subjects' homes (n=5 in total) on two consecutive daysfor each subject. Each subject was fully informed on the experimentalprocedures, and the study was approved by the Western InstitutionalReview Board (WIRB), Protocol Number 20130515.

Exclusion criterion: Subjects with severe lung disease (e.g., advancedchronic obstructive pulmonary disease, COPD) or those physically unableto provide breath samples into the SMART® mGC.

Breath samples were collected from five (5) adult (over the age of 21)study participants who were current smokers, and smoked in their homes.The study subjects will be identified in this report as SA-1, SA-2,SA-3, SA-4, and SA-5. The smoking frequency (i.e., self-reportedcigarette packs smoked per day) of these subjects, and the number ofactive smokers in the home for SA-1, SA-2, SA-3, SA-4, and SA-5 were0.5/2.5/2/1/1.5 and 4/2/1/3/2, respectively.

Each subject participated in the study for two (2) days. The studyvolunteers were randomized to smoke a single cigarette from each of thetwo (2) mentioned brands (i.e., Marlboro and Newport). A minimum of one(1) day was allowed between smoking the different cigarette brands. Noreplicate of a given cigarette brand was carried out for a givensubject. Participants were allowed food products and beverages adlibitum but were instructed not to take anything by mouth for 15 minutesprior to collection of the first breath sample and to refrain fromsmoking for a minimum of one (1) hour prior to providing the firstbreath sample.

A total of five (5) samples were collected using the SMART® mGCs duringeach home visit: one (1) room air sample and four (4) participant breathsamples. Paired baseline breath 2-butanone levels were established fromthe study participants prior to each smoking event by collecting a“blank” breath sample (T=−10 min). The study volunteers smoked therandomly assigned cigarette brand (i.e., either Marlboro or Newport) tocompletion, in their normal manner. Immediately after finishing thecigarette, each subject breathed into their designated SMART® mGC toprovide a time 0 (T=0 min) sample. Additional post-cigarette breathsamples were collected after 10 (T=10 min) and 20 minutes (T=20 min),thereafter. The study subjects had a minimum of one (1) day wait periodbetween smoking the different cigarette brands, after which the studyprotocol was repeated for each individual with the remaining cigarettebrand used in this interference screen (i.e., either Marlboro orNewport). During the wait period between the two study dates, thesubjects were not given any restrictions with regard to their regularsmoking habits.

Data Storage and Processing

All data was automatically uploaded and stored on a secured anddedicated Xhale server. First derivative of mGC sensor response versustime plots for the standards and breath samples were used to determine2-butanone peak heights and retention times.

Statistical Analyses

Exclusion of subject SA-5 from the statistical analysis: Breath samplescollected from study volunteer SA-5 resulted in a total loss of signalin the early part of the SMART® mGC chromatogram. This loss of signalwas considered a confounding variable for the purposes of evaluatingsmoking interferences and the data was therefore excluded from theanalysis. Although we could not confirm the exact cause of the breathVOC(s) that resulted in signal loss, this finding is consistent with theinterference observed at high concentrations of breath ethanol (e.g.,300,000 ppb ethanol). Participant SA-5 reported consuming approximately50 beers/week, and reported consuming an unspecified amount of beerapproximately two (2) hours prior to the beginning of the study. Thedata obtained from the indoor air of this study participant was includedin the analysis.

For the interference screen, data are expressed as mean±standarddeviation. Delta baseline was calculated as the mean change frombaseline in 2-butanone concentration (2-butanone concentration aftersmoking—2-butanone at baseline). 2-butanone concentrations below thelevel of detection (LoD) of the mGC (i.e., 5 ppb) were considered zerofor the delta baseline calculations. To determine the effect ofcigarette smoking on breath 2-butanone concentrations measured by theSMART® mGC, and whether significant differences exist between cigarettebrands (i.e., Marlboro vs. Newport) exist, the data were compared usingrepeated measures analysis of variance (ANOVA) (SigmaPlot 11.2, SystatSoftware, Inc., San Jose, Calif.). P-values <0.05 were consideredstatistically significant. Clinically significant interference fromsmoking cigarettes was defined as breath VOCs that changed the meanbaseline (prior to smoking) 2-butanone breath concentration by 5 ppb,the putative 2-butanone cutoff value supported by prior clinical mGCSMART® system performance studies (e.g., Example 3, Clinical Studies 1,2, and 3).

Results and Discussion

The SMART® Adherence System is used to confirm ingestion of medicationthat is associated with the AEM, 2-butanol. This is accomplished byevaluating the change in breath 2-butanone concentrations from baselineafter ingestion of 2-butanol. Cigarette smoking introduces a largenumber of VOCs in the breath of smokers. The presence of 2-butanone andother VOCs reported to be in cigarette smoke (e.g., ethyl acetate and3-methyl-1-butanol) that have retention times similar (100±5 seconds) tothat of 2-butanone on the SMART® mGC (Xhale Document No.: DR-0026), mayinterfere with the performance of the SMART® Adherence System and causeinaccurate 2-butanone results (i.e., false positives or falsenegatives). This study evaluated the effects of VOCs from cigarettesmoke present in 1) indoor air of homes of people who smoke in thehouse, and 2) breath samples from smokers at various times following asmoking event, on the apparent 2-butanone concentration measured by theSMART® mGC, in the absence of ingesting the AEM, 2-butanol.

The indoor air concentrations of 2-butanone were measured using theSMART mGCs in each home, on two separate occasions (one air sample perhome visit). The mean 2-butanone levels were measured to be below theLoD (<5 ppb) in four of the five homes tested (Homes 1, 2, 3 and 5).Indoor air from Home 4 had the highest mean concentration of 2-butanonemeasured by the SMART® mGC and was 6.9 ppb, which is slightly higherthan the LoD.

The baseline breath levels (i.e., prior to smoking) of 2-butanonemeasured by the SMART® mGC in the breath of study participants (n=4)ranged between below LoD 5 ppb) and 254.7 ppb. Note: In another study(protocol: Example 3, Clinical Study 1), it should be noted thatalthough high breath levels (132.4, 238.8, and 31.6 ppb) of background2-butanone were noted in subject 49, who was a smoker and admitted toconsuming a significant amount of alcoholic beverages in an ongoingbasis, he/she still responded favorably to the ingestion of the AEM,2-butanol, by generating large increases in breath 2-butanoneconcentrations above these higher than normal baseline levels. Thebaseline 2-butanone breath concentrations for participants SA-2 and SA-4were below the LoD for both study visits. The remaining study subjectsshowed large interpersonal variability in their baseline breath (i.e.prior to smoking the study cigarette) 2-butanone concentrations measuredduring the two home visits. The baseline breath 2-butanoneconcentrations measured for SA-1 were 5 ppb during the first home visitand 254.7 ppb during the second. In contrast, SA-3 had a relatively highbaseline breath 2-butanone concentration of 179.3 ppb during the firsthome visit, and 5 ppb during the second visit. Although both SA-2 andSA-3 had elevated 2-butanone levels in their baseline breath on the daythat they were given the Marlboro study cigarette, these concentrationswere measured prior to smoking, and therefor are independent of thecigarette brand used in this study.

During those visits with elevated baseline 2-butanone, subjects SA-1 andSA-3 showed decreases in their respective 2-butanone breathconcentrations with time that are consistent with the blood eliminationkinetics of 2-butanone (t1/2=49-96 minutes).⁴ These levels areapproximately 50 times greater than the 3-4 ppb of breath 2-butanonethat would be expected from the median blood 2-butanone concentration inthe general population (5.4 ppb) determined by the third National Healthand Nutrition Survey (NHANES III).⁶ This suggests the transient highbaseline levels of 2-butanone observed in these study subjects areincidental, and are not representative of the general population.

Breath samples were collected from each study subject at minutes priorto smoking (Baseline breath; T=−10 minutes), immediately (T=0 minutes),at 10 minutes (T=10 minutes) and 20 minutes (T=20 minutes), followingsmoking each cigarette brand (i.e., Newport and Marlboro). The SMART®mGC 1^(st) derivative chromatograms show that cigarette smoke introducedbreath VOCs with retention times on the SMART® mGC outside theinterference window for 2-butanone (i.e., 100±5 seconds). The SMART® mGCcan discriminate between 2-butanone and VOCs with retention timesgreater than ±5 seconds from 2-butanone. These VOCs are outside theinterference window, and do not interfere with the measurement of2-butanone by the SMART® mGC.

The change in baseline (pre-smoking) 2-butanone concentrationsregistered by the SMART® mGC for both cigarette brands at T=0, T=10 andT=20 minutes showed that no significant difference was observed ininterfering breath VOCs between the two cigarette brands (i.e., Newportand Marlboro) screened. Smoking did not result in a change in the meanbaseline (i.e., pre-smoking) 2-butanone level 5 ppb at any of the timepoints following the smoking event.

CONCLUSIONS

This study evaluated VOCs associated with smoking two commonly usedcigarette brands (i.e., Newport and Marlboro), for potentialinterference with SMART® mGC System performance. The kinetics of thesepotential interferents in human breath were evaluated in support of aplan to mitigate the risk of inaccurate SMART® mGC System results thatmay be associated with smoking.

VOCs present in the home environments had minimal effects on the2-butanone concentration measured by the SMART® mGC. Only one of thefive (1/5) homes resulted in mean indoor air 2-butanone concentrationsabove 5 ppb (mean concentration 6.9 ppb). The presence ofsmoking-derived VOCs, and the kinetics of potential interferents inhuman breath associated with smoking events was evaluated in studysubjects following use of Newport and Marlboro cigarettes. Smoking didnot result in a clinically significant change (i.e., 5 ppb) from thebaseline breath 2-butanone concentration on the SMART® mGC.

It appears that any potential risk of inaccurate 2-butanone results inhuman breath from smoking can be adequately mitigated by 1) collecting abaseline breath sample prior to ingestion of the AEM, 2-butanol, and 2)having a 15 minute wait period from smoking, before a subject breathsample is given. This finding is consistent with the results of clinicalstudies (Example 3: Clinical Study 1, 2, and 3) investigating theperformance of the SMART® mGC System, which demonstrated favorableperformance, even in subject populations enriched with participantshaving a significant smoking history.

REFERENCES

-   [1] Centers for Disease Control and Prevention. Current Cigarette    Smoking Among Adults—United States, 2011. Morbidity and Mortality    Weekly Report 2012; 61(44):889-94-   [2] Polzin, G. M., Kosa-Mains, R., Ashley, D. L., Watson, C. H.    Analysis of Volatile Organic Compounds in Mainstream Cigarette    Smoke, Environ. Sci. Technol. 2007, 41, 1297-1302.-   [3] “Toxic Volatile Organic Compounds in Environmental Tobacco    Smoke: Emission Factors for Modeling Exposures of California    Populations” by Lawrence Berkeley Laboratory under the sponsorship    of the California Air Resources Board. May 1994.    http://www.arb.ca.gov/research/apr/past/a133-186.pdf-   [4] Tobacco Brand Preferences. Center for Disease Control and    Prevention http://www.cdc.gov/tobacco/data    statistics/fact_sheets/tobacco_industry/brand preference/[5]-   [5] Lab data reference, SMART Logbook No. 8, pages 15-24—Document on    file at Xhale, Inc., Gainesville, Fla.-   [6] Churchill, J. E., Ashley, D. L., Kaye, W. E. Recent Chemical    Exposures and Blood Volatile Organic Compound Levels in a Large    Population Based Sample. Arch. Environ. Health. 2001, 56(2),    156-166.

Interference Study 3: Consumer Products

SUMMARY

The current study screened specific consumer products, based onknowledge of their flavorant content, that could potentially interferewith the performance of the 2-butanone-based SMART® mini-gaschromatographs (mGC) System. This could occur by the consumer productsproviding an additional breath source of 2-butanone and/or of anon-2-butanone VOC with a SMART® mGC retention time similar to that of2-butanone (100±5 sec). In the SMART® mGC System, the breath marker,2-butanone, is generated and detected (as change from baselineconcentration) in human breath by the mGC after ingesting the AEM,2-butanol.

The effects of fifteen different consumer goods, including fruits(banana), drinks (fruit drinks, coffee), candies, and health products(toothpaste, cough drops) on apparent 2-butanone breath levels measuredon the SMART® mGC were studied in four volunteer study participantsusing a cross over design. Each consumer product was kept in the mouthfor 30 seconds, then expectorated. 2-butanone concentrations weremeasured in baseline breath (i.e., in the absence of consumer product)and at various time intervals after the products were expectorated.Breath samples collected immediately (0 min), 10 min and 15 min, afterthe consumer goods were eliminated from the mouth, showed that 10/15(67%), 3/15 (20%), and 0/11 (0%) products caused an increase in baseline(pre-consumer product) 2-butanone levels 5 ppb, respectively. Thisfinding is consistent with the results of clinical studies (Example 3:Clinical Studies 1, 2, and 3) investigating the performance of theSMART® mGC System, which demonstrated favorable performance, even insubject populations who ingested food and drank liquids ad libitum butwere nothing per orum (NPO) 15 min or longer prior to study initiation(provision of baseline breath sample). Taken together, these resultssuggest two findings: 1) most foods will not interfere with theperformance of the SMART® mGC system, and/or 2) any potentialinterfering VOC is adequately cleared from the mouth within the 15 minNPO window.

INTRODUCTION

Natural and synthetic flavorants present in consumer products maycontain volatile organic compounds (VOCs) that can interfere with theperformance of the 2-butanone-based SMART® mGC System. This could occurby the consumer products providing an additional breath source of2-butanone and/or of a non-2-butanone VOC with a SMART® mGC retentiontime similar to that of 2-butanone (100±5 sec). In the SMART® mGCSystem, the breath marker, 2-butanone, is generated and detected (aschange from baseline concentration) in human breath by the mGC afteringesting the AEM, 2-butanol. Based on our knowledge of what VOCs have asimilar retention time to 2-butanone and the flavorant composition offood, the purpose of this study was to perform a screen of variousfoods, drinks, and other consumer goods, which would be the most likelyto interference with the system by introducing interfering VOCs in themouth, and subsequently alter the concentrations of 2-butanone measuredin exhaled breath (i.e., in the absence of the AEM. The kinetics ofthese potential interferents in human breath were evaluated in supportof a plan to mitigate the risk of inaccurate 2-butanone results on theSMART® System. We previously demonstrated that foods (e.g., yogurt,cheddar cheese, black tea, tomatoes), which are known to contain thehighest endogenous content of the AEM, 2-butanol and the breath marker,2-butanone, do not appear to interfere with the SMART® mGC System, evenwhen rapidly ingested in large quantities.¹

Materials and Methods

Test Articles and Formulations

Four SMART® mGCs from Xhale Inc. were used for this study. One mGCdevice was used per person, and the instruments were randomly assignedto each individual. The mGCs used had serial numbers 100112120001,100112120003, 100113010007, and 100113010010. These units are identifiedas 212-01, 212-03, 301-07, and 301-10 in this report.

1-L Tedlar gas sampling bags were purchased from SKC Inc. (Eighty Four,Pa.). Each bag was used only once. A single 10.0 mL Hamilton gas-tightsyringe (Model Number 1010, Fisher Scientific part number 14-815-183)was used for the dilution of the 2-butanone gas standard.

2-Butanone standards were created by diluting a primary NIST certified10 ppm 2-butanone gas standard in dry nitrogen (Matheson Tri-Gas MICROMAT 58 Item Number GMT2677977TH, Lot Number 109-26-07599, ExpirationDate 5/11/14) into 1-L Tedlar bags containing blank breath. The 15consumer products selected for this study were all purchased from PublixSupermarket in Gainesville Fla. the day before the study began (exceptfor the Arcor Strawberry Buds Candy, which was supplied by the studysponsor). The products tested, and the abbreviation used in this reportwere as follows: Fruit (banana), Health products (Cologne Totaltoothpaste; Fresh Burst mouthwash, triple soothing strawberry coughdrop); Candies (various types of gum, flavored hard candies, jelly fruitslices, and cinnamon breath mints), and Beverages (Nestle coffee withcreamer, Arizona fruit drink).

Butanone Standard Creation and Analysis

Dilution of a NIST-certified 2-butanone gas standard into Tedlar gassampling bags containing a blank breath sample was performed to create astandard curve at four concentrations (0, 10, 100, and 1000 ppb).Standard curves for 2-butanone were created for each of the four SMART®mGCs used in this study.

Investigational Plan

Each subject was fully informed on the experimental procedures, and thestudy was approved by the Institutional Review Board (IRB), Universityof Florida. Exclusion Criteria: Subjects found physically unable toprovide breath samples.

Breath samples were analyzed using the individual mGCs from four (4)adult study participants. The participants were instructed not toconsume alcoholic beverages the day before the study, and not to eat,drink or smoke for 15 minutes prior to the beginning of the study.

The study was carried out in two phases. The first phase screened 15consumer products, to evaluate interference of mouth VOCs with theSMART® mGC immediately after, and 10 minutes following each product. Abaseline 2-butanone level was established for each subject by analyzinga “blank” breath sample 10 minutes before placing each consumer productin their mouth. To maximize the concentrations of mouth VOC, thesubjects kept each consumer product in their mouth and mixed it aroundfor 30 seconds, and then expectorated. Immediately after each productwas eliminated from the mouth, the study subjects breathed into theirdesignated SMART® mGC to provide a time 0 (T=0 min) sample. A secondpost-consumer product breath sample was collected 10 minutes later (T=10min). To prevent carry-over of potential interferents between theproducts, the study participants rinsed their mouths thoroughly withwater after each item tested, and waited a minimum of 15 minutes beforerepeating the procedure for the remaining products.

For the second phase of the study, nine products were chosen from theinitial screen to evaluate the presence of interfering VOCs in thebreath after 15 minutes (T=15 minutes) from the time the items wereexpectorated. The study protocol was the same as described for theinitial screen, with the exception of the time intervals used to collectthe post-consumer product breath samples. In this study, theparticipants waited for 15 minutes after eliminating each consumerproduct from their mouth before providing the post-consumer productbreath sample. The consumer products chosen for the second phase oftesting were identified in the initial screen as having the highestlevels of potential interferents.

Clinical significant interference from consumer products was defined asbreath VOCs that changed the mean baseline (pre-consumer product)2-butanone breath concentration by 5 ppb.

Data Storage and Processing

All data was automatically stored to the Xhale secured servers. Firstderivative plots for the standards and breath samples were imported intoMicrosoft Excel (Redmond, Wash.), and the peaks and retention times weredetermined for each compound.

Statistical Analyses

For the interference screen, data are expressed as mean±standarddeviation. Delta baseline was calculated as the mean change frombaseline 2-butanone (mGC SMART® 2-butanone concentration after consumerproduct—mGC SMART® 2-butanone at baseline) in parts per billion (ppb).2-butanone concentrations below the LoD (i.e., 5 ppb) were consideredzero for the delta baseline calculations. Descriptive statistics of thedata were calculated using SigmaPlot 11.2, Systat Software, Inc. (SanJose, Calif.).

Results and Discussion

The SMART® Adherence System is used to confirm ingestion of a medicationthat is associated with the AEM, 2-butanol. This is accomplished byevaluating the change in breath 2-butanone concentrations from baselinelevels, after ingestion of the AEM, 2-butanol. Eating/drinking foods,and/or using healthcare products that contain either 2-butanone, or anon-2-butanone VOC with a retention time similar to 2-butanone on theSMART® mGC (100±5 seconds) may result in inaccurate results (i.e., falsepositive or false negative results). Four VOCs (methyl acrylate, ethylacetate, 3-butene-1-ol and cyclohexane) were previously identified ashaving similar retention times on the SMART® mGC as 2-butanone. One ofthese VOCs, ethyl acetate, elutes within 1 second of 2-butanone, and isa flavoring agent found in food or health products. This acetate isnaturally occurring in fruits,³ and it is a direct food additive (40 CFR180.910) used as fruit essence in food items.⁴ The interference screenof consumer goods was done using products known to contain natural orsynthetic flavoring agents that would be likely to interfere with theSMART® mGC System.

The baseline levels of 2-butanone measured in the breath of studyparticipants (n=4) before each consumer product were below the LoD (<5ppb) for both the first and second part of the study. In the first phaseof the study 15 consumer products were used to determine their potentialto interfere with the SMART® mGC system. The mean concentrations ofapparent 2-butanone measured by the SMART mGC in exhaled breathimmediately was measured after each product was expelled from the mouth.The change in baseline (pre-consumer product) 2-butanone concentrationsregistered by the SMART® mGC for each consumer product at T=0 minuteswas determined. At T=0 minutes, as illustrated, 10 of the 15 (67%)products tested resulted in a change in the mean baseline (pre-consumerproduct) 2-butanone level 5 ppb. These foods produce a clinicallysignificant interference on the SMART® mGC at T=0 minutes. Of the 15consumer goods, the banana caused the SMART® mGC to register the highestchange in the mean breath concentration of 2-butanone (1481.3±296.0ppb).

The breath samples taken 10 minutes after the consumer products wereexpectorated from the mouth show that the 2-butanone concentrationmeasured by the SMART® mGC were below the LoD (5 ppb) of the SMART® mGCfor 11 of the 15 (73%) products. The changes from baseline breath(pre-consumer product) 2-butanone concentrations registered for eachconsumer product at T=10 minutes was determined. Three of the 15 (3/15)products resulted in a change in the mean baseline (pre-consumerproduct) 2-butanone level 5 ppb at T=10 minutes. The SMART® mGC 1stderivative chromatograms show that the banana also introduced breathVOCs (e.g., ethanol) with early retention times on the SMART® mGC(retention times in the 20-60 second range), that do not interfere withthe 2-butanone measurement. This was observed with other consumerproducts as well (data not shown). The presence of these additionalbreath VOCs do not have clinical significance for the SMART® mGC, butcan be qualitatively assessed if interference from consumer products issuspected. At T=10 minutes, three of the 15 (20%) consumer goods producea clinically significant interference on the SMART® mGC at 10 minutes.

The second phase of the study was done to determine if a 15 minute waitafter consumer products are expelled from the mouth, is adequate for themeasured 2-butanone levels to return to baseline. The concentrations of2-butanone in the baseline breath, and 15 minutes post-consumer productwere determined. At T=15 minutes, the 2-butanone concentrations inbreath were below the LoD (5 ppb) of the SMART® mGC, for all theconsumer products. This indicates that within 15 minutes of eliminatingthe products from the mouth, the breath 2-butanone response on theSMART® mGC returns to baseline levels. The consumer products do notproduce a clinically significant interference on the SMART® mGC 15minutes after they are eliminated from the mouth.

The potential interference of Listerine mouthwash on the SMART® mGC atT=0 and T=10 minutes was not determined in this study. Listerine did notproduce a measurable change in the mean baseline (pre-consumer product)2-butanone levels at either sampling times. However, a qualitativeassessment of the SMART® mGC 1st derivative chromatograms from thebreaths samples obtained at T=0 and T=10 minutes, indicates thatListerine, which contains 20% v. ethanol, may cause a negative bias in2-butanone measurements at these time points. Ethanol interference withthe SMART® mGC was evaluated, and is presented in Example 3:Interference Study 4. A qualitative assessment of Listerine mouthwashinterference with the 2-butanone measurement on the SMART® mGC was alsodone using the 1^(st) derivative chromatograms from T=15 minute. Thebreath 2-butanone response on the SMART mGC returns to baseline(pre-consumer product) within 15 minutes after the time the mouthwash iseliminated from the mouth.

The persistence of the interfering breath VOCs (i.e., apparent2-butanone concentrations >5 ppb above baseline) from consumer productswas determined. The first phase of the study showed that at T=0 minutes,10/15 (67%) and at T=10 minutes 3/15 (20%) of the products result in achange in the mean baseline (pre-consumer product) 2-butanone level 5ppb. The second phase of the study indicates that 2-butanone response onthe SMART® mGC returns to baseline within 15 minutes after expectoratingthe consumer products from the mouth.

CONCLUSIONS

Food, drink, or other consumer products may contain VOCs that have thepotential to adversely impact the performance of the 2-butanone-basedSMART® Type 1 (mGC) System. This could occur by introducing additional2-butanone and/or non-2-butanone VOCs to human breath that have asimilar SMART® mGC retention time to 2-butanone (100±5 sec). However,within 15 minutes from the time the products are eliminated from themouth, the breath 2-butanone concentration response on the SMART® mGCreturns to baseline (pre-consumer product). Therefore, the risk ofinaccurate 2-butanone results in human breath associated with thestudied consumer products can be adequately mitigated by 1) collecting abaseline breath sample prior to ingestion of the AEM, 2-butanol, and 2)having a 15 minute wait period from use of food or drink (note: thisdoes not refer to the ingestion of standard liquids commonly used toingest medications such as water, tea, coffee, etc.), before a subjectbreath sample is given. This finding is consistent with the favorableSMART® mGC System performance results of clinical studies (Example 3:Clinical Studies 1-3), where subjects were allowed to be fed ad libitumprior to enrollment but kept nothing per orum (NPO) 15 min prior toproviding the baseline breath sample.

REFERENCES

-   [1] Morey T E, Wasdo S, Wishin J, Quinn B, Booth M, Gonzalez D,    Derendorf H, McGorray S P, Simoni J, Melker R J, Dennis D M: Oral    Adherence Monitoring Using a Breath Test to Supplement Highly Active    Antiretroviral Therapy, AIDS Behav, 17(1), 298-306, 2013.-   [2] Lab data reference, Xhale Logbook BPQ 2012-1, pages 45-48 and    page 53. Document on file at Xhale, Inc., Gainesville, Fla.-   [3] http://www.epa.gov/opprd001/inerts/ethyl_amyl_acetate. pdf-   [4] http://www.epa.gov/iris/subst/0157.htm

Interference Study 4: Ethanol

SUMMARY

Interference testing was performed to determine the effect of increasingconcentrations of ethanol in the breath on the 2-butanone-based SMART®mGC System. Test samples were prepared using breath samples (n=5 studyvolunteers) spiked with 50 ppb 2-butanol, and ethanol concentrations of0, 30,000, 100,000, and 300,000 ppb. The effects of ethanol weredetermined by comparing the 2-butanone retention time and SMART® mGCresponse (2-butanone 1st derivative peak height) in control samples(i.e., no ethanol) and test samples containing the variousconcentrations of ethanol.

High concentrations of ethanol in breath did not affect the retentiontime of 2-butanone on the SMART® mGC but did reduce the mGC response to2-butanone. For example, using the upper limit of the 95% confidenceinterval (per CLSI EP7-A2 guidance), ethanol at 30,000, 100,000, and300,000 ppb in breath reduced the SMART mGC response to ppb 2-butanonealone by 50%, 64%, and 74%, respectively. However, it should be notedthat this reduction in apparent 2-butanone breath concentration byethanol should not be a significant issue with regard to SMART® mGCperformance, because the majority of unique subjects in clinicalstudies, who ingest the AEM, 2-butanol, generate increases in breath2-butanone concentration above baseline values much greater than 5 ppb(Example 3: Clinical Studies 1-3). This finding is consistent with theresults of clinical studies (Example 3: Clinical Studies 1-3)investigating the performance of the SMART® mGC System, whichdemonstrated favorable performance, even in subject populations enrichedwith participants having a significant alcohol drinking history. Last,the potential negative impact of high concentrations of ethanol on themeasurement of 2-butanone in breath can be rapidly recognized, assessed,and mitigated by examining the “front end” of the SMART® mGCchromatogram.

INTRODUCTION

Ethanol is a volatile organic compound (VOC) that is typically found inthe exhaled breath in trace amounts (low parts per billion or ppb), as aresult of endogenous processes (e.g., sugar metabolism in the colon). Aprevious study by Morey et al., (2012)[1] showed that endogenous ethanolpresent in the breath matrix did not interfere with the ability of theSMART® mGC to measure 2-butanone, the breath marker used in the SMART®System. Ingesting drink products containing ethanol (e.g., alcoholicbeverages) may increase the concentrations of breath ethanol to levelsthat are several orders of magnitudes greater than endogenous levels.For example, the legal limit of intoxication in most jurisidcations ofthe United States is a blood alcohol content (BAC) of 0.08% (80 mgethanol per dL blood), which corresponds to a breath alcoholconcentration (BrAC) of approximately 200 ppm (200,000 ppb).

The objective of this study was to evaluate the potential of elevatedethanol concentrations to interfere with the ability of the SMART® mGCto measure 2-butanone in human breath. To determine the degree ofinterference from ethanol, the mGC response (i.e., 2-butanone peakheight) and 2-butanone retention time, were measured in breath samples“spiked” with 50 ppb 2-butanone in the presence of progressivelyincreasing concentrations of ethanol (0, 30,000, 100,000, and 300,000ppb). The 50 ppb concentration of 2-butanone was selected for theinterference test because 1) it reflects a typical lower endconcentration of 2-butanone that appears in breath after ingestion of atypical dose of 2-butanol (i.e., 20 and 40 mg) (Example 3: ClinicalStudy 1-3), 2) it is close to the anticipated yes/no cutoff which willbe used to determine medication adherence, and 3) based on prior devicevalidation testing, it can be reliably used to measure a potentialdecrease in the mGC response.

Materials and Methods

Test Articles and Formulations

Interference testing was conducted using four (4) SMART® mGCs from XhaleInc. The mGCs used had serial numbers 100113010009, 100113010010,100113010011, and 100113010015, and are identified as 10009, 10010,10011, and 10015, respectively, in this report.

1-L Tedlar gas sampling bags were purchased from SKC Inc. (Eighty Four,Pa.). Each bag was used only once. A single 10.0 mL Hamilton gas-tightsyringe (Model Number 1010, Fisher Scientific part number 14-815-183)was used for the dilution of the 2-butanone gas standard.

The 200 proof anhydrous (>99.5%) ethanol used in this study waspurchased from Sigma-Aldrich (part number 459835-100ML, Batch 54096BM).This neat standard was diluted in deionized water (DI) to make workingsolutions for injection into the Tedlar bags containing blank breathspiked with 2-butanone. 2-Butanone standards were created by diluting aprimary NIST certified 10 ppm 2-butanone gas standard in dry nitrogen(Matheson Tri-Gas MICRO MAT 58 Item Number GMT2677977TH, Lot Number109-26-07599, Expiration Date 5/11/14) into 1-L Tedlar bags containingblank breath.

2-Butanone Standard Creation and Analysis

The four SMART® mGCs used in this study were calibrated at the NanoscaleResearch Facility of the University of Florida. Dilution of aNIST-certified 2-butanone gas standard into Tedlar gas sampling bagscontaining a blank breath sample was performed to create a calibrationcurve at four concentrations (0, 10, 25, and 50 ppb). Standard curvesfor 2-butanone were created on each of the four SMART® mGC.

Investigational Plan

The methodology in this study was developed using guidance from theClinical and Laboratory Standards Institute (CLSI). Interference Testingin Clinical Chemistry; Approved Guideline, EP7-A2.

Ethanol interference with 2-butanone measurement was evaluated usingpaired-difference testing, by measuring the SMART® mGC response of2-butanone (50 ppb) in the presence of increasing ethanol concentrations(30,000, 100,000, and 300,000 ppb), in spiked human breath samples.

The studies were performed in the Nanoscale Research Laboratory at theUniversity of Florida. Each subject was fully informed on theexperimental procedures, and the study was approved by the WesternInstitutional Review Board (WIRB) protocol 20130515.

Breath samples were collected from five (5) adult study participants. Inorder to provide baseline breath samples relatively free of VOCsincluding ethanol, participants were instructed not to consume alcoholicbeverages for one day (24 h) and not to eat, drink or smoke for 15minutes prior to their study visits. Each study volunteer provided sixseparate breath samples into individual 1-L Tedlar gas sampling bagsover a period of 30 minutes (up to five minute break periods wereallowed between breath samples).

Exclusion Criteria: Subjects found to have a high level of ethanol intheir breath, or those physically unable to exhale 1-L breath samplesinto the Tedlar gas sampling bags.

After collection, the five breath samples from each participant weregiven a subject identifier (A, B C, D or E) and labeled individually asVOC 1 to 5. All samples were allowed to equilibrate overnight before theaddition of 2-butanone or ethanol. After initial equilibration, bags VOC2, VOC 3, VOC 4 and VOC 5 from each subject were spiked with 50 ppb of2-butanone. Bags VOC 3, VOC 4 and VOC 5 from each subject wereadditionally spiked with 1 μL aliquots of aqueous ethanol standards tomake breath samples containing 30,000, 100,000, and 300,000 ppb ethanol.Sample preparation of stock solutions, 2-butanone samples and testsamples (i.e., containing three concentrations of ethanol) is listedbelow:

50 Ppb 2-Butanone

The 50 ppb 2-butanone concentration was obtained by diluting 5 cc of the10 ppm of a NIST-certified 2-butanone gas standard, into Tedlar gassampling bags containing 1-L blank breath sample. For the testing pool,neat ethanol was first diluted into DI water, then 1 μL aliquots wereinjected into 1-L of blank breath to make the ethanol standards used inthis study as follows:

30,000 Ppb Ethanol

70.5 μL of ethanol (55.7 μg) was diluted to 1.00 mL with water toproduce a 55.7 μg/mL bag spiking solution. One μl of spiking solutionwas injected into an equilibrated 1-L Tedlar bag containing blank humanbreath to produce a 30,000 ppb (30 ppm) breath ethanol standard. 55.7 μgof ethanol/1 L of breath=1308 μg/24.789 L of breath

100,000 Ppb Ethanol

235 μL of ethanol (186 μg) was diluted to 1.00 mL with water to producea 186 μg/mL bag spiking solution. One μl of spiking solution wasinjected into an equilibrated 1-L Tedlar bag containing blank humanbreath to produce a 100,000 ppb (100 ppm) breath ethanol standard. 186μg of ethanol/1 L of breath=4611 μg/24.789 L of breath

300,000 Ppb Ethanol

705 μL of ethanol (557 μg) was diluted to 1.00 mL with water to producea 557 μg/mL bag spiking solution. One μl of spiking solution wasinjected into an equilibrated 1-L Tedlar bag containing blank humanbreath to produce a 300,000 ppb (30 ppm) breath ethanol standard. 557 μgof ethanol/1 L of breath=13807 μg/24.789 L of breath

Sample bags were allowed to equilibrate for two hours after addition ofthe final spiking component before analysis. The SMART® mGC 2-butanoneresponse was evaluated in the spiked study bags (5 breath samples pertest concentration), using each of the four (4) study SMART® mGC units.

Data Storage and Processing

All data was automatically stored to the Xhale secured servers. Firstderivative plots for the standards were imported into Microsoft Excel(Redmond, Wash.), and the peaks and retention times were determined foreach compound.

Statistical Analyses

The degree of ethanol interference with the SMART® mGCs was calculatedfor each concentration of ethanol using Equation 1. For each device,results are expressed as mean percent interference at each concentrationof ethanol (n=5 breath samples).

100×(mGC

response

_((Interferent+Analyte))−

mGC response

_((Analyte)))/

mGC response

_((Analyte))   (1)

where: the interferent is Ethanol

-   -   the analyte is 2-butanol (50 ppb)

Statistical analysis of ethanol interference on mGC response to 50 ppb2-butanone was carried out using one way analysis of variance (ANOVA)(SigmaPlot 11.2, Systat Software, Inc., San Jose, Calif.). P-values<0.05 were considered statistically significant. The upper limit of the95% confidence interval (CI) for the interference effect was calculatedacross the four devices (n=4 devices). Clinical significant interferencefrom ethanol was defined as a change in the SMART® mGC 2-butanolresponse of ≧20%.

Results and Discussion

The interfering effects from elevated concentrations of ethanol weredetermined on the ability of the SMART® mGC to detect 2-butanone at twolevels using guidance from CLSI EP7: 1) response to 2-butanone asmeasured by the 1^(st) derivative, and 2) 2-butanone retention times.Summary of the 2-butanone retention times and mGC response (i.e., 1^(st)Derivative peak height) for control (i.e., 50 ppb 2-butanone withoutethanol) and test samples (i.e., 50 ppb 2-butanol with ethanol at thespecified concentrations) were created. Ethanol did not affect theretention time of 2-butanone on the SMART® mGC. In contrast, allconcentrations of ethanol tested in this study caused a statisticallysignificant decrease (P<0.05) in the SMART® mGC response to 50 ppb2-butanone relative to control.

The percent interference observed for each of the four SMART® mGCs, thatresulted from ethanol (30,000, 100,000, and 300,000 ppb) added to breathsamples containing 50 ppb of 2-butanone was determined. At the lowestconcentration of ethanol (30,000 ppb), the mean 2-butanone response onthe mGCs ranged between ˜15.3% (mGC#10010) and -48.6% (mGC #10009)relative to control (i.e., 50 ppb 2-butanone without ethanol). At thehighest concentration of ethanol (300,000 ppb) the mean mGC 2-butanoneresponse showed a decrease of up to ˜70.3% (mGC#10010). Increasingethanol concentrations resulted in a negative bias in the SMART® mGCresponse to 50 ppb 2-butanone on all the SMART® mGCs.

The overall interference caused by ethanol (30,000, 100,000, and 300,000ppb) when added to breath samples containing 50 ppb 2-butanone wascalculated as the upper limit of the 95% confidence interval (per CLSIEP7-A2 guidance) using the mean percent interference observed for eachdevice (n=4 devices). Interference from ethanol with the 2-butanoneresponse on the SMART® mGCs was calculated to be −50% (30,000 ppb), −64%(100,000 ppb), and −74% (300,000 ppb).

An ethanol concentration of 300,000 ppb can temporarily saturate thedetector, and because of 1st derivative artifacts, appears as a loss ofsignal at the front-end of the chromatogram (this typically occursbetween 25 and 40 s). However, since 2-butanone elutes much later thanethanol (approximately 70 seconds later), although the SMART® mGCresponse for 50 ppb 2-butanone is significantly decreased (by up to 50%at 30,000 ppb ethanol), a 2-butanone peak remains distinguishable evenat the highest ethanol concentration (i.e., 300,000 ppb) studied. Thepresence of a distinguishable ethanol peak, or the apparent loss mGCresponse observed in the “front end” of the mGC chromatogram can bequalitatively assessed, and be used as an indicator of potential ethanolinterference with the SMART® mGC System.

CONCLUSIONS

High concentrations of ethanol in breath can potentially interfere withthe performance of the SMART® System by decreasing the 2-butanoneresponse on the SMART® mGC. Using the upper limit of the 95% confidenceinterval (per CLSI EP7-A2 guidance), ethanol at 30,000, 100,000, and300,000 ppb in breath reduced the SMART® mGC response to ppb 2-butanonealone by 50%, 64%, and 74%, respectively. In contrast, the retentiontime of 2-butanone on the SMART® mGC was not affected by the presence ofhigh concentrations of ethanol in breath.

The risk of inaccurate 2-butanone results in human breath (i.e., falsenegative results) that is associated with high breath concentrations ofethanol can be mitigated by at least two factors. First, this reductionin apparent 2-butanone breath concentration by ethanol will not be asignificant issue with regard to SMART® mGC performance, because themajority of subjects (i.e., 98.4% positive response rate in 185subjects) in clinical studies, who ingest the AEM, 2-butanol, generateincreases in breath 2-butanone concentration above baseline values muchgreater than 5 ppb (Example 3: Clinical Studies 1-3). This finding isconsistent with the results of clinical studies (Example 3: ClinicalStudies 1-3) investigating the performance of the SMART® mGC System,which demonstrated favorable performance, even in subject populationsenriched with participants having a significant alcohol drinkinghistory. Second, the potential negative impact of high concentrations ofethanol on the measurement of 2-butanone in breath can be rapidlyrecognized and mitigated by a qualitative assessment of the “front end”of the SMART® mGC chromatogram. Specifically, if a 2-butanone peak isnot found on the SMART® mGC after ingesting the AEM, 2-butanol, and themGC chromatogram indicates the presence of breath ethanol or signalloss, it would indicate that the presence of an interferent (i.e.,alcohol) may have caused the breath concentration of 2-butanone to befalsely low.

REFERENCES

-   [1] Morey et al, Oral Adherence Monitoring Using a Breath Test to    Supplement Highly Active Antiretroviral Therapy, AIDS Behav    17(1):298-306. 2013-   [a] Lab data reference, SMART Logbook No. 8, pages 13-14—Document on    file at Xhale, Inc., Gainesville, Fla.-   [2] Wang, C., Yin, L., Xhang, L., Xiang, D., Gao, R. Metal Oxide Gas    Sensors: Sensitivity and Influencing Factors. Sensors 2010, 10,    2088-2106.-   [3] Barsan, N., Weimar, U., Understanding the Fundamental Principles    of Metal oxide based gas sensors; the example of CO sensing with    SnO2 sensors in the presence of humidity. J. Phys. Condens. Matter    15 (2003) R813-R839.-   [4] Logen, B. K., Sistefano, S. Ethanol Content of Various Foods and    Soft Drinks and their Potential for Interference with a    Breath-Alcohol Test. Journal of Analytical Toxicology, Vol. 22,    May/June 1998, 181-183.-   [5] Phillips M, Greenberg J: Endogenous breath ethanol    concentrations in abstinent alcohol abusers and normals Alcohol    5(3):263-265, 1988.

Overall Conclusions from Interference Studies

In conclusion the results of the four potential interferent studiesindicate that the impact of potential interferents can be mitigiated oreven eliminated by using a baseline breath sample to detect anybackground EDIMs and correct for it and/or simply waiting a period oftime for volatiles to clear from the mouth.

Furthermore, two alternate designs of the SMART® Adherence System can beused to markedly mitigate and even eliminate these potentialinterference to adherence system function:

In a first embodiment to overcome interference, an adherence system isimplemented that uses a Type I SMART® device to measure the simultaneousappearance in human breath of two or more EDIMs in human breath afteringestion of a medication labeled with two (or more) different AEMs.Example 4a illustrates and enables this approach. The appearance ofmultiple EDIMs after ingesting a medication labeled with more than oneAEM will be so highly distinctive that it will not only eliminate mostinterferences, but it may well eliminate the need for a baseline breathsample in order to accurately detect adherence in the SMART® AdherenceSystem.

Second, an adherence system that uses a Type II SMART® device (e.g.,infrared based detector that measures e.g. deuterated EDIMs) will haveno environmental or endogenous interferents. It is essentially free ofinterferents and should not require a baseline breath sample. Likewise,the latter approach using a Type 2 device (e.g., mid-IR) makes ittechnologically much easier to design and implement a SMART systems usedfor intermediate medication adherence monitoring (IMAM) and chronicmedication adherence monitoring (CMAM), because an AEM like isopropylalcohol will generate acetone, which has a breath half life of severalhours to approximately a day. Because humans have significant amounts ofendogenous acetone in breath, this limits the utility of this approachusing a Type I device (mGC-MOS) since it would react to the endogenousacetone. In contrast, a Type 2 device (e.g., mid-IR), would detectdeuterated acetone which would be generated from deuterated isopropylalcohol. Furthermore, mid=IR system can be extremely sensitive (pptrange) to measuring deuterated water.

Example 4a Illustrations of how Multiple AEMs can be Employed in theSMART® Adherence System to Generate Different EDIMs in Human Breath thatare Measured by a Type 1 SMART® Device

The use of more than AEM in the SMART Adherence System to generate morethan one EDIM can have a number of advantages: 1) the simultaneousappearance of multiple EDIMs in breath will essentially eliminatepotential interferents (e.g., environmental, endogenous)

This example describes how two different AEMs (2-butanol and2-pentanone) can be used to generate two different EDIMs with similarhalf lives in human breath that are measured by a Type 1 SMART® device.

Appearance of 2-butanone and 2-pentanone as the EDIMs in fasting humansafter ingestion of a hard gel capsule containing an AEM formulationcomposed of 60 mg 2-butanol and 60 mg 2-pentanone. Note in the case of2-butanol, the EDIM is a metabolite of the AEM, whereas in the case of2-pentanone, it serves the role as the AEM and the EDIM (comes out inbreath intact and not metabolized). The data shows that althoughinter-individual variability of EDIM appearance is greater thanintra-individual variability, it does not affect the ability of the Type1 device-based SMART® Adherence System to assess adherence. Likewise, itappears that early appearance of the EDIM in the breath following oralingestion is primarily dependent on absorption of the AEM through thestomach mucosa and not enzymatic conversion of the secondary alcohol,2-butanol, to 2-butanone.

SUMMARY

A work- or residence-based, self-administered medication adherencemonitoring system using exhaled breath is useful to identify and reduceclinical trial nonadherence. We studied the inter- and intra-individualvariability for the exhalation of two adherence markers (2-butanone,2-pentanone) in healthy subjects (n=5) with six replicates followingoral consumption of encapsulated 2-butanol and 2-pentanone.Minimal-to-no intra-individual variability was observed forone-compartment pharmacokinetic parameters. Some inter-individualvariability was noted for half-life, maximal concentrations, andarea-under-the-curve estimates. Intra- or inter-individual variation inthe time to achieve threshold concentrations of 2-butanone or2-pentanone to signal adherence was not observed. ROC analysis revealedpositive and negative predictive values near unity for breath samplingtimes >5 min and assumed adherence rates of 50-90%. The concurrentexhalation of 2-butanone and 2-pentanone indicates that enzymaticcatalysis of 2-butanol to 2-butanone is not a rate limiting step of thesystem. We conclude that even with mild inter-individual variability,the system signals adherence from time points 10-60 min.

INTRODUCTION

Adherence to prescribed medication regimens is an important,uncontrolled source of variation in clinical trials spanning manytherapeutic classes [1-5]. Peck attests to this fact by noting thatunknown adherence behavior by subjects is the single largest determinantof variation in biological responses following theophyllineadministration [6]. This result is largely due to propagation oferroneous assumptions that subjects actually were adherent intocalculations of pharmacokinetics (PK), pharmacodynamics (PD), andprimary clinical endpoints. These ideas are borne into reality with suchclinical trials as the Preexposure Prophylaxis Initiative (iPrEx)wherein subjects with >90% adherence had a 73% risk reduction for HIVacquisition with oral tenofovir disoproxil fumarate and emtricitabinetherapy, but subjects with <90% adherence had only a 21% benefit [1, 7].Similarly, Woldu and colleagues discovered that nonadherence was “ . . .a common and significant source of treatment nonresponse . . . ” for 190therapy-resistant, depressed adolescents contemplating suicide andbelieved by investigators to be receiving selective serotonin reuptakeinhibitors [8]. Additionally, whereas great attention is focused on drugformulation to exert rigid tolerances on chemistry manufacturing andcontrol (CMC), minimal-to-no effort occurs to even measure adherence.Moreover, subject adherence is the last, potentially measurable and/orgovernable event prior to uncontrollable PK and PD properties that areunique to each subject. For these reasons, we assert that subjectadherence is important to measure in clinical trials and previouslysuggested a new method to achieve this aim [9-11].α

This novel technique entails co-packaging an innocuous, chemical taggantwith an oral medication. The taggant (or its metabolite) may appear inexhaled breath after absorption by the gastrointestinal mucosa. Forexample, we demonstrated in a feasibility study that 2-butanone israpidly exhaled (5-15 min) after subjects swallowed encapsulated2-butanol [9]. Furthermore, 2-butanone can be readily measured by ahome- or work-based, portable, self-administered, HIPAA-compliant,miniature gas chromatographic (mGC) device that conveys this informationto a central data repository for review by study coordinators or healthcare personnel [10]. Moreover, this technique can be used for non-oralroutes of delivery, such as vaginal or rectal administration [10].Herein, we sought to determine the intra- and inter-individualvariability associated with this technique to monitor oral adherencewith respect to exhalation of 2-butanone, a metabolite of 2-butanol (ataggant incorporated into a capsule) catalyzed by αα-alcoholdehydrogenase (ADH), an enzymatic isoform not subject to ethnicvariations as are other variants of ADH [12-14]. Additionally, because2-butanol essentially serves as an inactive “prodrug” for adherenceverification, we also added 2-pentanone that we hypothesize is exhaledwithout the need for metabolism due to its physical characteristics.

Methods

Test Materials. 2-butanol (60 mg), 2-pentanone (60 mg), and inertL-carvone (30 mg) were inserted within a hard gel capsule (size 3,LiCaps®, Capsugel, Greenwood, S.C.). 2-butanol was purchased from PentaManufacturing Company (Fairfield, N.J.). 2-pentanol and L-carvone werepurchased from Sigma-Aldrich (St. Louis, Mo.). Each capsule constituenthad a unique role. 2-butanol was used as a taggant that is metabolizedto produce 2-butanone, a volatile marker that appears in breath.2-pentanone was also used as a taggant, but we hypothesized that itsinherent volatility would allow exhalation without need for metabolism.L-carvone tastes like spearmint and was used as a flavor mask. Allcapsules were filled on the day of the experiment.

Subject Enrollment and Protocol. This protocol (20100140) was approvedby the Western Institutional Review Board (Olympia, Wash.). Healthysubjects (n=5) aged >18 years of age and of either sex were recruited.Informed, written consent was obtained from all enrolled subjects. Thestudy consisted of a single experimental limb with six replicates persubject. Therefore, for five subjects with six replicated experiments, atotal of 30 studies were performed. Subjects were free to eat ad libprior to participation in the study. At commencement of the experiments,subjects provided a baseline breath sample (designated time 0 min) intothe mGC for analysis as described subsequently. Then, subjects orallyconsumed the previously detailed capsule with 177 mL (6 ounces) ofwater. Following ingestion, we observed the breath concentration-timerelationships of 2-butanone and 2-pentanone by collecting single-breathsamples at times 5, 10, 15, 20, 30, 45, and 60 min. After 60 min, theexperiment was concluded. After at least one day's respite, subjectsreturned for replicate experiments on five additional occasions.

2-Butanone and 2-Pentanone Measurement in Human Breath by mGC. Breathspecimens were analyzed “real-time” to measure the concentrations of2-butanone and 2-pentanone using the mGC (Xhale, Inc. Gainesville, Fla.)as previously described [9-11, 15]. In brief, a mouthpiece (FST®,Intoximeters, Inc., St. Louis, Mo.) was attached to the inlet of themGC. A 15 mL side-stream sample was aspirated during a single exhalationover 5 s through this inlet port to a room temperature concentrator trapcontaining 4 mg of Tenax TA (Sigma-Aldrich). The trap temperature wasthen increased to 130° C. and the volatile components transferred to a 5m by 0.53 mm internal diameter, metal clad, BAC-1 capillary column(Restek Corp, Bellefonte, Pa.) operated at 55° C. The effluent from thecolumn then flowed to the metal oxide detectors. Separation of themarkers from other volatile organic compounds present in the sample(i.e., acetone and isoprene) occurred in approximately 2 min. Data weretabulated and graphically presented over time to determine theconcentrations of 2-butanone and 2-pentanone in a given breath specimen.Data are reported in parts-per-billion (ppb) based on molar fractions toaccount for differing ambient atmospheric pressures and temperature[10]. A sample mGC chromatogram of a human breath sample followingingestion of the hard gel capsule containing 60 mg 2-butanol and 60 mg2-pentanone is shown in FIG. 75 a.

Data Analysis

Pharmacokinetic Analysis. Analyte breath concentrations reported as ppbwere converted to ng/mL by multiplying each concentration by themolecular weight of the respective molecule to allow proper functioningof conventional PK software for noncompartmental analysis (WinNonlin5.2, Pharsight Corporation, St. Louis, Mo.). Estimates were generatedfor the following PK parameters (abbreviation, unit): first-orderelimination rate constant (Lambda Z, min-1), half-life of elimination(Half-life, min), maximal drug concentration (C_(MAX), μg/mL), time atmaximal drug concentration (T_(MAX), min), area under concentrationversus time curve from zero to the last time point (AUC_(0-LAST),min*μg/mL), area under concentration versus time curve from zero toinfinity (AUC_(0-∞), min*μg/mL), percentage of area under concentrationversus time curve from zero to infinity, which is extrapolated fromAUC_(0-t) (% AUC Extrap) and the mean residence time (MRT, min). Thearea under concentration versus time curve (AUC) was calculated usingthe linear trapezoidal rule. All values are reported separately for eachsubject as mean±standard deviation following conversion from ng/mL toppb. For parameters obtained from the non-compartmental analysis, thecoefficient of variation across subjects was calculated for eachreplicate. Additionally, the threshold time (T_(Thresh)) was determinedthat represents the time for the breath concentration of 2-butanone or2-pentanone to exceed the detection concentration for mGC.

Receiver Operator Curves. ROC analysis was conducted using SigmaPlot12.3 (Systat Software, Inc., Chicago, Ill.). When modeling positive andnegative predictive values based on measured concentrations of2-butanone or 2-pentanone, we assumed pre-test probability rates of 50%,70%, and 90% for actual adherence.

Results

Each of the five subjects successfully completed a total of sixreplicated studies. Demographically, subjects were aged 47±5 years with3 men and 2 women, all of non-Hispanic, white race self-identification.Their mean body mass and mean height was 89±30 kg and 179±15 cm,respectively, for a calculated mean body mass index of 27±5 kg/m2. Noadverse events were reported or observed. For all 30 visits, 2-butanoneand 2-pentanone appeared in breath as measured by the mGC. The overallconcentration-time plots for 2-butanone and 2-pentanone are shown inFIG. 75b and demonstrate the similarity of these relationships for bothexhaled markers. The PK parameter estimates from the noncompartmentalanalysis for 2-butanone and 2-pentanone are noted in the following Table4a-I:

TABLE 4a-I Exhaled 2-butanone and 2-pentanone pharmacokinetic parameterestimates for noncompartmental analysis for human subjects (n = 5) withsix replicates for each subject. T_(Thresh) represents the time for thebreath concentration of 2-butanone or 2-pentanone to exceed thedetection concentration for the miniature gas chromatograph (mGC). Dataexpressed as mean (standard deviation) for 30 observations (5 subjectswith 6 replicates per subject). Parameter 2-butanone 2-pentanone PT_(Thresh) (min) 6.2 (2.2) 5.7 (1.7) 0.08 LAMBDA_Z(min⁻¹) 0.036 (0.013)0.033 (0.012) <0.01 Half-life (min) 216 (8.0) 23.7 (9.1) 0.03 C_(MAX)(ppb) 1376 (820) 1424 (741) 0.25 T_(MAX) (min) 17.8 (8.8) 14.8 (8.0)<0.01 AUC_(0-LAST) (min · ppb) 41820 (27104) 45614 (25196) <0.01AUC₀₋₈(min · ppb) 51190 (32170) 58214 (32308) <0.01 % AUC Extrap 17.9(8.4) 19.7 (8.9) 0.10 MRT_(0-last) (min) 25.7 (4.8) 25.4 (4.1) 0.32

Both exhaled markers appeared quickly in breath with similar T_(Thresh)values of approximately 5-6 min. Comparing 2-butanone and 2-pentanone,significant differences of modest magnitude were observed for LAMDA_Z,half-life, T_(MAX), AUC_(0-LAST), and AUC_(0-∞). In most cases,2-butanone and 2-pentanone concentrations could be quantified in breathwithin 5 min post-ingestion and were still detectable at 60 min, thescheduled termination for each study.

Inter-subject Variability. To discover the possible presence ofinter-subject variability, the mean concentrations for 2-butanone and2-pentanone for each time point were plotted by subject (FIG. 75c ). Asexpected, time markedly affected the concentration of both exhaledmarkers (P<0.01). Additionally, the particular subject significantlyaffected the concentration-time relationships (P<0.01). Calculated PKparameters for 2-butanone and 2-pentanone are shown in Tables 4a-II and4a-III, respectively.

TABLE 4a-II Inter-individual, exhaled 2-butanone pharmacokineticparameter estimates for noncompartmental analysis for human subjects (n= 5) with six replicates for each subject. T_(Thresh) represents thetime for the breath concentration of 2-butanone to exceed the detectionconcentration for the miniature gas chromatograph (mGC). Data expressedas mean (standard deviation). SUB- SUB- SUB- SUB- SUB- JECT JECT JECTJECT JECT Parameter 1 2 3 4 5 P T_(Thresh) 6.7 5.0 5.8 5.8 7.5 0.38(min) (2.6) (0.0) (2.0) (2.0) (2.7) LAMBDA_Z 0.033 0.034 0.024 0.0430.046 <0.01 (min⁻¹) (0.008) (0.005) (0.008) (0.010) (0.017) Half-life22.6 20.6 30.7 16.4 17.5 <0.01 (min) (6.1) (3.2) (9.0) (3.4) (8.9)C_(MAX) 1297 1926 1021 2238 398 <0.01 (ppb) (451) (240) (710) (709)(258) T_(MAX) 21.7 20.8 11.7 18.3 16.7 0.37 (min) (10.3) (4.9) (10.8)(8.2) (7.5) AUC_(0-LAST) 45298 66466 19608 68147 9581 <0.01 (min · ppb)(16577) (5169) (11599) (18758) (7641) AUC_(0-∞) 59106 82628 25623 7759310999 <0.01 (min · ppb) (19881) (10044) (15159) (19556) (8576) % AUC22.6 19.2 23.2 12.6 12.0 0.02 Extrap (11.0) (3.5) (4.7) (3.3) (10.5)MRT_(0-Last) 29.3 27.7 22.7 26.2 22.9 0.06 (min) (5.9) (1.6) (5.5) (3.2)(3.6)

TABLE 4a-III Inter-individual, exhaled 2-pentanone pharmacokineticparameter estimates for noncompartmental analysis for human subjects (n= 5) with six replicates for each subject. T_(Thresh) represents thetime for the breath concentration of 2-pentanone to exceed the detectionconcentration for the miniature gas chromatograph (mGC). Data expressedas mean (standard deviation) SUB- SUB- SUB- SUB- SUB- JECT JECT JECTJECT JECT Parameter 1 2 3 4 5 P T_(Thresh) 5.8 5.0 5.8 5.0 6.7 0.43(min) (2.0) (0.0) (2.0) (0.0) (2.6) LAMBDA_Z 0.029 0.030 0.023 0.0400.042 <0.01 (min⁻¹) (0.007) (0.004) (0.009) (0.08) (0.015) Half-life24.8 23.2 34.6 17.9 17.8 <0.01 (min) (5.6) (2.6) (13.3) (3.5) (5.2)C_(MAX) 1328 1850 1134 2072 536 <0.01 (ppb) (396) (180) (859) (697)(290) T_(MAX) 20.0 15.8 10.0 15.0 13.3 0.34 (min) (9.5) (4.9) (10.0)(7.7) (6.1) AUC_(0-Last) 49094 68008 31020 66164 13783 <0.01 (min · ppb)(17665) (6249) (18913) (17202) (9108) AUC_(0-∞) 65626 87240 45947 7645015806 <0.01 (min · ppb) (21989) (9622) (34360) (19188) (10707) % AUC24.5 21.9 27.1 13.6 11.1 <0.01 Extrap (9.7) (2.6) (9.1) (2.7) (5.5)MRT_(0-last) 28.4 27.3 23.7 25.6 22.2 0.40 (min) (4.9) (1.4) (4.6) (2.6)(3.4)

Although significant differences were observed for several PKparameters, we did not observe significant inter-subject variabilitywith respect to TThresh for either 2-butaone (P=0.38) or 2-pentanone(P=0.43).

Intra-subject Variability. To determine the degree of intra-subjectvariability in exhalation of the breath markers, we re-indexed theconcentration-time relationships for 2-butanone and 2-pentanol byreplicated group 1-6 (FIG. 75d ). For all subjects, these relationshipswere significantly affected by time as expected, but the replicategroups had no overall effect for either 2-butanone (P=0.32) or2-pentanone (P=0.12). PK parameter estimates were calculated for2-butanone (Table 4a-IV) and 2-pentanone (Table 4a-V) by replicategroups.

TABLE 4a-IV Intra-individual, exhaled 2-butanone pharmacokineticparameter estimates for noncompartmental analysis for human subjects (n= 5) with six replicates for each subject. T_(Thresh) represents thetime for the breath concentration of 2-butanone to exceed the detectionconcentration for the miniature gas chromatograph (mGC). Data expressedas mean (standard deviation) for the same 5 subjects in each replicate.REPLI- REPLI- REPLI- REPLI- REPLI- REPLI- Parameter CATE 1 CATE 2 CATE 3CATE 4 CATE 5 CATE 6 P T_(Thresh) 5.0 7.0 6.0 6.0 7.0 6.0 0.72 (min)(0.0) (2.7) (2.2) (2.2) (2.7) (2.2) LAMBDA_Z 0.036 0.033 0.048 0.0380.026 0.037 0.04 (min⁻¹) (0.011) (0.013) (0.019) (0.013) (0.007) (0.004)Half-life 21.1 24.1 16.4 21.2 27.7 18.9 0.09 (min) (7.0) (9.8) (5.7)(11.8) (6.7) (1.9) C_(MAX) 1793 1361 1175 1449 1227 1251 0.47 (ppb)(585) (502) (1089) (1259) (630) (887) T_(MAX) 15.0 21.0 17.0 19.0 15.020.0 0.85 (min) (7.1) (10.2) (9.7) (10.8) (10.6) (6.1) AUC_(0-LAST)53278 44693 36508 44467 37792 34184 0.19 (min · ppb) (26769) (19393)(33613) (38862) (26067) (24534) AUC_(0-∞) 64618 58070 42226 52141 49178340905 0.10 (min · ppb) (33892) (24341) (38250) (43170) (32325 (29441) %AUC 41.3 47.3 31.3 37.3 54.6 39.2 0.07 Extrap (13.8) (16.2) (16.2)(20.9) (11.3) (7.1) MRT_(0-last) 24.4 28.3 24.8 23.9 26.6 26.5 0.60(min) (4.0) (6.2) (4.2) (4.6) (6.4) (3.6)

TABLE 4a-V Intra-individual, exhaled 2-pentanone pharmacokineticparameter estimates for noncompartmental analysis for human subjects (n= 5) with six replicates for each subject. T_(Thresh) represents thetime for the breath concentration of 2-pentanone to exceed the detectionconcentration for the miniature gas chromatograph (mGC). Data expressedas mean (standard deviation) for the same 5 subjects in each replicate.REPLI- REPLI- REPLI- REPLI- REPLI- REPLI- Parameter CATE 1 CATE 2 CATE 3CATE 4 CATE 5 CATE 6 P T_(Thresh) 5.0 7.0 5.0 6.0 5.0 6.0 0.37 (min)(0.0) (2.7) (0.0) (2.2) (0.0) (2.2) LAMBDA_Z 0.030 0.032 0.042 0.0330.027 0.034 0.17 (min⁻¹) (0.012) (0.015) (0.018) (0.008) (0.004) (0.004)Half-life 27.5 26.1 18.7 22.7 26.1 20.9 0.32 (min) (15.2) (12.6) (6.7)(8.3) (3.9) (2.6) C_(MAX) 1885 1472 1253 1519 1297 1119 0.31 (ppb) (558)(493) (9038) (1152) (595) (694) T_(MAX) 15.0 15.0 12.0 12 15.0 20 0.67(min) (7.1) (10.0) (7.6) (7.6) (10.6) (6.1) AUC_(0-LAST) 61008 4978840006 46918 41780 34184 0.05 (min · ppb) (22007) (16350) (31881) (34942)(24960) (21188) AUC_(0-∞) 81227 66524 48111 56515 54688 42218 0.03 (min· ppb) (31128) (23396) (37965) (40544) (32234) (32234) % AUC 22.8 23.014.8 16.6 22.2 18.5 0.23 Extrap (12.6) (11.7) (8.7) (7.0) (7.7) (3.4)MRT_(0-Last) 25.2 27.3 24.1 24.2 25.3 26.5 0.73 (min) (2.5) (5.1) (5.1)(3.6) (5.4) (3.2)

In contrast, to Table 4a-II and 4a-III, only LAMDA_Z attainedstatistical significance for 2-butanol and AUC0-∞ for 2-pentanone.Importantly, T_(Thresh) for 2-butanone (P=0.72) or 2-pentanone (P=0.37)did not significantly differ between replicates and had a range of 5-7min. 3 Additionally, we plotted the all concentration values of2-butanone (n=240) against that for concurrently collected 2-pentanone(FIG. 75e ). The regressed line demonstrated a very high coefficient ofdetermination (r2=0.93) and a slope near identity (0.99±0.02).

ROC Analysis. We performed ROC determinations to understand the abilityof this system to predict adherence. Values for ROC analysis are notedin Table 4a-VI for both 2-butanone and 2-pentanone.

TABLE 4a-VI Receiver operator curve (ROC) data for exhaled 2-butanoneand 2- pentanone from human subjects (n = 5 subjects with 6 replicates/subject) after orally consuming encapsulated 2-butanol and 2-pentanone.Data shown are ROC areas with parenthetical 95% confidence intervals.P_(0.50) is the Type I error risk that a ROC area for a given time pointis different from a ROC area of 0.50. 2-butanone 2-pentanone Time (min)ROC Area P_(0·50) ROC Area P_(0·50) 5 0.88 (0.81-0.96) <0.01 0.93(0.87-0.99) <0.01 10 1.00 (1.00-1.00) <0.01 1.00 (1.00-1.00) <0.01 151.00 (1.00-1.00) <0.01 1.00 (1.00-1.00) <0.01 20 1.00 (1.00-1.00) <0.011.00 (1.00-1.00) <0.01 30 1.00 (1.00-1.00) <0.01 1.00 (1.00-1.00) <0.0145 1.00 (1.00-1.00) <0.01 1.00 (1.00-1.00) <0.01 60 1.00 (1.00-1.00)<0.01 1.00 (1.00-1.00) <0.01

The ROC areas for both taggants escalated rapidly to unity during thefirst 10 min after capsule ingestion and remained sustained up to 60min, the conclusion of the study. Additional sensitivity and specificitydata at suggested cut-off concentrations are tabulated for 2-butanoneand 2-pentanone in tables 4a-VII and 4a-VIII, respectively.

TABLE 4a-VII Sensitivity and specificity analysis detailing accuracyover 5-60 min with suggested “cut off” concentrations for exhaled2-butanone from human subjects (n = 5 subjects with 6replicates/subject) after orally consuming encapsulated 2-butanol and2-pentanone. Cut-off represents the concentration of 2-butanone abovewhich a subject is identified as adherent. Parenthetical values are 95%confidence intervals. Abbreviations: PPV, positive predictive value;NPV, negative predictive value. Predictive values were based onsensitivity and specificity for an assumed rate of adherence asspecified in the table. Assumed Adherence Time Cut-off Rate (%) PPV/NPV(min) (ppb) Sensitivity Specificity 50 70 90  5 20.9 0.77 1.00 1.00/1.00/ 1.00/ (0.58-0.90) (0.88-1.00) 0.81 0.65 0.32 10 15.1 1.00 1.001.00/ 1.00/ 1.00/ (0.88-1.00) (0.88-1.00) 1.00 1.00 1.00 15 29.8 1.001.00 1.00/ 1.00/ 1.00/ (0.88-1.00) (0.88-1.00) 1.00 1.00 1.00 20 40.11.00 1.00 1.00/ 1.00/ 1.00/ (0.88-1.00) (0.88-1.00) 1.00 1.00 1.00 309.2 1.00 1.00 1.00/ 1.00/ 1.00/ (0.88-1.00) (0.88-1.00) 1.00 1.00 1.0045 2.1 1.00 1.00 1.00/ 1.00/ 1.00/ (0.88-1.00) (0.88-1.00) 1.00 1.001.00 60 2.0 1.00 1.00 1.00/ 1.00/ 1.00/ (0.88-1.00) (0.88-1.00) 1.001.00 1.00

TABLE 4a-VIII Sensitivity and specificity analysis detailing accuracyover 5-60 min with suggested “cut off” concentrations for exhaled2-pentanone from human subjects (n = 5 subjects with 6replicates/subject) after orally consuming encapsulated 2-butanol and2-pentanone. Cut-off represents the concentration of 2-butanone abovewhich a subject is identified as adherent. Parenthetical values are 95%confidence intervals. Abbreviations: PPV, positive predictive value;NPV, negative predictive value. Predictive values were based onsensitivity and specificity for an assumed rate of adherence asspecified in the table. Assumed Adherence Time Cut-off Rate (%) PPV/NPV(min) (ppb) Sensitivity Specificity 50 70 90  5 29.1 0.87 1.00 1.00/1.00/ 1.00/ (0.69-0.96) (0.88-1.00) 0.88 0.76 0.45 10 56.5 1.00 1.001.00/ 1.00/ 1.00/ (0.88-1.00) (0.88-1.00) 1.00 1.00 1.00 15 59.1 1.001.00 1.00/ 1.00/ 1.00/ (0.88-1.00) (0.88-1.00) 1.00 1.00 1.00 20 57.51.00 1.00 1.00/ 1.00/ 1.00/ (0.88-1.00) (0.88-1.00) 1.00 1.00 1.00 3027.6 1.00 1.00 1.00/ 1.00/ 1.00/ (0.88-1.00) (0.88-1.00) 1.00 1.00 1.0045 10.0 1.00 1.00 1.00/ 1.00/ 1.00/ (0.88-1.00) (0.88-1.00) 1.00 1.001.00 60  3.5 1.00 1.00 1.00/ 1.00/ 1.00/ (0.88-1.00) (0.88-1.00) 1.001.00 1.00

Also, calculated positive and negative predictive values are noted forboth taggants in these tables for pre-test probability adherence ratesof 50%, 70%, and 90%, the adherence rate from the iPrEx study [1, 7].From times 10-60 min, positive and negative predictive values were 1.00and 1.00, respectively, for adherence rates of 50%-90%.

DISCUSSION

In this study, 2-butanone and 2-pentanone concentrations could bemeasured by mGC in every subject's and every replicate's breathfollowing ingestion of encapsulated 2-butanol and 2-pentanone. In mostcases, detectable concentrations were observed as early as 5 min and inall cases by 10 min after ingestion. As illustrated in FIGS. 75c and 75dand in the tabulated PK data, we observed more inter-individualvariability compared to intra-individual variability. We suggest thatthe majority of inter-individual variability is may be due tobioavailability of 2-butanol and 2-pentanone. To our knowledge,minimal-to-no research has been reported describing the bioavailabilityof these compounds in humans, although some animal work has beenpublished. Dietz and colleagues fed 2-butanol to rats to understand thiscompound's metabolism [16]. They reported that approximately 97% ofingested 2-butanol is metabolized to 2-butanone, that the major site ofmetabolism is the liver, and that transformation rate is dependent onliver blood perfusion. In the absence of detailed human data for2-butanol or 2-pentanone, we hypothesize that consideration of anotheralcohol's (i.e., ethanol) PK may be instructive since the PK of ethanolhas been well studied due to its legal implications. In a review,Norberg and colleagues noted that gastrointestinal absorption is animportant parameter of ethanol PK and that the “ . . . major factorgoverning the absorption rate of ethanol is whether the drink is takenon an empty stomach (overnight fast) or together with or after a meal”with minor factors including meal composition, liver blood flow, andothers [17]. Notwithstanding, although we did not control for feedingstate in the present study, in another investigation we demonstratedthat fasting or consumption of a high fat meal did not affect productionof 2-butanone in humans [9]. The total dose of ethanol in human studiesis, however, orders of magnitude greater than that for 2-butanol.Assuming 10 g of ethanol in a standard drink and reiterating that theauthors used 0.060 g of 2-butanol, a single ethanol beverage contains166,667-fold more alcohol. The experimental model used hereinunfortunately does not allow control for other factors affectingabsorption across the gastrointestinal mucosa or differing bloodperfusion from gastric veins to the portal venous circulation of theliver.

Irregular elimination of 2-butanone and 2-pentanone by exhalationbetween subjects may also lead to the inter-individual variability.Again looking to ethanol for guidance, Lindberg and Grubb concluded thatthe volume of respiratory dead space in a particular subject markedlyaffects the arterial blood-to-breath concentrations of another exhaledethanol [18]. Likewise, variations in voluntary breath patterns maycause some PK differences even though careful instructions and practiceexhalation were provided to each subject in the present report. That is,Boshier and colleagues observed that different subject respiratorymaneuvers (e.g., hyperventilation, breath holding) significantlymodified the exhaled concentrations of ethanol, methanol, and acetone[19]. For these additional reasons, PK may vary both between individualsalthough little variation was observed within an individual subject over6 replicated studies.

In the broader context, however, the magnitude of these inter- andintra-individual variations must be placed in the clinical context ofthe required concentrations to measure adherence when deployed to studysubjects. That is, the primary purpose of addition of the taggants2-butanol and 2-pentanone was to facilitate monitoring of oral adherenceusing a portable, self-administered, HIPAA-compliant mGC. To that end,PK parameters must be considered in the context of the capabilities ofthe mGC and clinical trial design. Notwithstanding the observed PKinter-individual variability, 2-pentanone and 2-butanone concentrationscould be readily quantified in all 30 visits by the mGC. Considering thelower concentration limits of quantification for the mGC ofapproximately 1.0 ppb for both 2-butanone and 2-pentanone, the observedC_(MAX) ranges for 2-butanone (398-2,238 ppb) and 2-pentanone (536-2,072ppb) were more than suitable to measure these analytes. In fact, suchlarge concentrations emanating from the lungs with T_(MAX) values of10-22 min suggest that subjects may be able to provide breath specimensto the mGC in a much shorter time frame after consuming a capsule.Likewise, the rapid rate of rise for both exhaled compounds toT_(Thresh) values of 5-6 min alludes to the possibility of measuring thecompounds sooner after ingestion of the capsule. That is, the window toprovide a breath sample is currently 5-60 min. This period may bebroadened to include some values <5 min and perhaps longer than 60 min.Rapid detection of these molecules in breath following oraladministration allows for flexibility and convenience for subjectsactually providing breath specimens.

Additionally, we determined that the variability did not impact overallassessments of capsule consumption using ROC analysis. At every timepoint after consumption based on R_(3.50) values, this system wasmarkedly better than guessing. Similarly, the positive and negativepredictive values for a variety of pre-test probabilities were unity fortime points after 5 min for both 2-butanone and/or 2-pentanone. Thenegative predictive value data reported herein are improved compared toearlier studies of 2-butanone wherein these values were 0.31-0.89 fortime points 30-60 min after ingestion [9]. We believe that the 50%increase in 2-butanol mass from 40 mg to 60 mg likely accounted for thisimprovement in the last half of study observations and suggests thatstudy design will inform decision making about the dose of adherencemarker for an particular clinical trial. The negative predictive valuedata from time points 0-20 min were very similar and appeared to notchange with increased 2-butanol mass whereas the positive predictivevalue data were similar for all time points. Recently, van der Stratenand colleagues observed that a breath test for use of vaginal placementof tenofovir placebo gel or lubricated condoms was “ . . . 100% accurate. . . ” although these investigators used esters of 2-butanol and2-pentanol [11]. To date, there are no previous reports of exhaled2-pentanone for review and comparison to the data presented herein.Therefore, the system provided adequate adherence signals even withintra- and inter-individual variability, differing doses betweenstudies, and both oral and vaginal routes (rectal administration remainsto be examined).

Three limitations warrant mention when considering this data that may bevaluable to those skilled in the art when practicing the presentinvention and implementing the SMART® system. (A) Resolution andduration of sampling: Earlier and more frequent sampling at times <5 minenable fine discrimination of the minimum latency before a breath samplecan be provided to measure adherence. Increasing the duration ofsampling allows sufficient decrements in exhaled marker concentrations(which were approximately 100-1,000 ppb at 60 min) to improve errorrevolving around the AUC PK parameters. (B) Differences in ambienttemperature and pressure require us to report exhaled gas parameters asppb based on molar fractions [10]. In this study, we provided a constantmass (60 mg) of 2-butanol (molar mass: 74.13 g/mol) and 2-pentanol(molar mass: 86.13 g/mol) which leads to differing molar doses becausethese two compounds have different molecular weights. That is, weprovided 13.8% more 2-butanol than 2-pentanol based on molar dosingwhich complicates data interpretation. Notwithstanding, the exhalationof both these markers was approximately the same based on the slope ofidentity noted in FIG. 75e , which indicates two findings: 1) the PKparameters of the AEMs and their EDIMs are similar (not surprising giventheir structural similarities and close molecular weights), and 2) theprocesses of absorption of the AEMs (2-butanol and 2-pentanone) from thegastrointestinal tract (e.g., stomach) are very rapid and enzymaticconversion of 2-butanol to 2-butanone is not rate limiting. This findingof a slope of 1 for equal mass dosing (60 mg) of 2-butanol and2-pentanone, imply that different drugs and/or dosage forms could beeffective labeled by varying the ratio of 2-butanol to 2-pentanone. Forexample, because their structures and molecular characteristics are sosimilar, it is expected that their PK parameters should be very similar.This finding is consistent with the slope of 1 found in FIG. 75e . Thus,it would be expected based on this disclosure that an orallyadministered AEM formulation containing a 2-butanol: 2-pentanone doseratio of 10:1, 3:1, 1:1, 1:3, 1:10 would yield an 2-butanone:2-pentanone EDIM ratio of 10, 3, 1, 0.33, and 0.10, respectively. Thiswould manifest as a graph as shown in FIG. 75e with 2-butanone (y axis):2-pentanone (x axis) slopes of 10, 3, 1, 0.33, and 0.10, respectively.(C) The subjects reported in this pilot project were healthy and of amodest magnitude number. Greater numbers of subjects and cohorts withHIV/AIDS and other diseases increases the data available for applicationof this system to these particular populations.

CONCLUSIONS

The results of this pilot study demonstrate that 2-butanone and2-pentanone can be detected in breath following oral administration. Forthe purposes of measuring oral adherence within the context of mGC useand relatively large exhaled concentrations of 2-butanone and2-pentanone, the variability in PK appears to have a negligible effecton adherence signals.

REFERENCES

-   1. Grant R M, Lama J R, Anderson P L, et al. Preexposure    chemoprophylaxis for HIV prevention in men who have sex with men. N    Engl J Med. 2010; 363:2587-99.-   2. Hogg R S, Heath K, Bangsberg D, et al. Intermittent use of    triple-combination therapy is predictive of mortality at baseline    and after 1 year of follow-up. AIDS. 2002; 16:1051-8.-   3. Lo R V, Teal V, Localio A R, et al. Relationship between    adherence to hepatitis C virus therapy and virologic outcomes: a    cohort study. Ann Intern Med. 2011; 155:353-60.-   4. Rolnick S, Pawloski P, Bruzek R, et al. PS2-32: Barriers and    facilitators for medication adherence. Clin Med Res. 2011; 9:157.-   5. Allen N E, Sherrington C, Suriyarachchi G D, et al. Exercise and    motor training in people with Parkinson's disease: a systematic    review of participant characteristics, intervention delivery,    retention rates, adherence, and adverse events in clinical trials.    Parkinsons Dis. 2012; 2012:854328.-   6. Harter J G, Peck C C. Chronobiology. Suggestions for integrating    it into drug development. Ann N Y Acad Sci. 1991; 618:563-71.-   7. Interim Guidance: Preexposure Prophylaxis for the Prevention of    HIV Infection in Men Who Have Sex with Men. MMWR Morb Mortal Wkly    Rep. 2011; 60:65-8.-   8. Woldu H, Porta G, Goldstein T, et al. Pharmacokinetically and    clinician-determined adherence to an antidepressant regimen and    clinical outcome in the TORDIA trial. J Am Acad Child Adolesc    Psychiatry. 2011; 50:490-8.-   9. Morey T E, Booth M, Wasdo S, et al. Oral Adherence Monitoring    Using a Breath Test to Supplement Highly Active Antiretroviral    Therapy. AIDS Behay. 2012; [Epub ahead of print].-   10. Morey T E, Wasdo S, Wishin J, et al. Feasibility of a breath    test for monitoring adherence to vaginal administration of    anti-retroviral microbicide gels. J Clin Pharmacol. 2012; [Epub    ahead of print].-   11. van der Straten A, Cheng H, Wasdo S, et al. A novel breath test    to directly measure use of vaginal gel and condoms. AIDS Behay.    2012; In Press.-   12. Reddy B, Reddy A, Nagaraja T, et al. Single nucleotide    polymorphisms of the alcohol dehydrogenase genes among the 28 caste    and tribal populations of India. Int J Hum Genet. 2006; 6:309-16.-   13. Bosron W F, Magnes L J, Li T K. Human liver alcohol    dehydrogenase: ADH Indianapolis results from genetic polymorphism at    the ADH2 gene locus. Biochem Genet.-   1983; 21:735-44.-   14. Edenberg H, Bosron W F. Alcohol Dehydrogenases, In: Guengerich F    (editor). Biotransormation Vol. 3, Comprehensive Toxicology. New    York, N.Y.: Pergamon Press; 1997. pp. 119-31.-   15. Morey T, Booth M M, Prather R A, et al. Measurement of ethanol    in gaseous breath using a miniature gas chromatograph. J Anal    Toxicol. 2011; 35:134-42.-   16. Dietz F K, Rodriguez-Giaxola M, Traiger G J, et al.    Pharmacokinetics of 2-butanol and its metabolites in the rat. J    Pharmacokinet Biopharm. 1981; 9:553-76.-   17. Norberg A, Jones A W, Hahn R G, et al. Role of variability in    explaining ethanol pharmacokinetics: research and forensic    applications. Clin Pharmacokinet. 2003; 42:1-31.-   18. Lindberg L, Grubb D. Simultaneously recorded single-exhalation    profiles of ethanol, water vapour and CO(2) in humans: impact of    pharmacokinetic phases on ethanol airway exchange. J Breath Res.    2012; 6:036001.-   19. Boshier P R, Priest O H, Hanna G B, et al. Influence of    respiratory variables on the on-line detection of exhaled trace    gases by PTR-M S. Thorax. 2011; 66:919-20.-   20. Lindbom L, Pihlgren P, Jonsson E N. PsN-Toolkit—a collection of    computer intensive statistical methods for non-linear mixed effect    modeling using NONMEM. Comput Methods Programs Biomed. 2005;    79:241-57.-   21. Keizer R J, van B M, Beijnen J H, et al. Pirana and PCluster: a    modeling environment and cluster infrastructure for NONMEM. Comput    Methods Programs Biomed. 2011; 101:72-9.

Example 4b Illustration of how Two Different AEMs (2-Butanol and2-Isopropyl Alcohol) can be Used to Generate Two Different EDIMs withDifferent Half Lives in Human Breath that are Measured by a Type 1SMART® Device

A fasting subject ingested a soft gel containing an AEM formulationconsisting of 2-butanol (40 mg) and isopropyl alcohol (30 mg). Shown inPanel A of FIG. 76 is the 1^(st) derivative mGC response (proportionalto EDIM breath concentration) in a Type 1 SMART Device for acetone and2-butanone as a function of breath sampling times post ingestion of thecapsule.

Panel B depicts the change from baseline of the 1^(st) derivative mGCresponse. Time 0 is baseline (immediately before capsule is ingestedcontaining the two AEMs). Within 10 minutes, rises in the breathconcentration of both 2-butanone and acetone can be easily noted,peaking with a Tmax of 20 min. Note how the 2-butanone levels in breathbegin to fall after 20 min, whereas those of acetone slowly continue torise over the study period. These findings are consistent with a halflife of 2-butanone of about 45 minutes and a half life of acetoneranging from ˜6.4 hrs (see Example 26 herein) to 17-27 hrs (previouslyreported with isopropanol poisoning (see, e.g., Jones, J. Anal Toxicol(January-February 2000) 24 (1) 8-10. hrs). Thus, not only will the useof combined EDIMs essentially eliminate potential interferents, it mayeliminate the need for a baseline breath to be taken, and it enhancescertainty when practicing this invention in the AMAM, IMAM, and,especially, CMAM modes.

The use of an AEM, like IPA, which generates a longer half life EDIM,like acetone, allows for much longer look back periods in terms ofadherence behavior and makes chronic medication adherence monitoring(CMAM) viable (see Example 26 for further details).

The mean acetone concentration in human breath has been found to rangefrom 293 to 870 ppb over a 30 day period (Diskin A M et al: Physiol Meas24:107-119, 2003). Note that the background level of 2-butanone is verylow whereas, as expected, the human breath contains a significantquantity of acetone as a result of lipid metabolism.

Example 5 Breath Kinetics of Exhaled d6-Acetone and d7-IsopropanolFollowing the Topical Application of d8-Isopropanol in a Carbomer Gel

Transdermal:

240 mg of d8-isopropanol was mixed with 3 mL of a carbomer-based aloegel. This gel was applied to an approximately 20 cm² area of the innerleft forearm and covered with a Tagaderm occlusive dressing. To furtherreduce the permeability of the dressing, the transparent section wascovered with a small section of teldar polymer prior to use.

Oral:

Either 100 mg d8-isopropanol or 20 mg d6 isopropanol was deliveredorally. For oral dosing, 100 or 20 mg of neat d8-isopropanol were placedin a size 4 licap and the licap was swallowed along with 60-100 mL ofwater. Following administration of d8-isopropanol, exhaled breath wasmonitored in real time for the presence of d6-acetone and d7-isopropanolusing the Orbitrap LCMS.

Results:

Following application or ingestion, d6-acetone and d7-isopropanol levelswere monitored in exhaled breath samples using the LTQ-LCMS. Single fullbreath samples were administered directly into the modified ESI sourceat 5 min intervals for ˜4 hours. The ESI source was operated in positiveion mode. A 0.2% NH₄OH:water mobile phase was introduced into the sourceat a flow rate of 0.1 mL/min during sampling to produce ammonium adductsof the analytes of interest. As can be seen in FIG. 69, by 15 minutespost-ingestion of either 100 mg d8-IPA (left hand axis) or 20 mg d8-IPA(right hand axis), D6-acetone levels in the exhaled breath began tolevel out and remain at maximum levels for several hours. By contrast,d-8 isopropanol delivered transdermally (right hand axis) resulted inmuch slower kinetics of appearance of d-6 acetone in the exhaled breath,with a maximum concentration still not achieved by 200 minutes postapplication.

These data demonstrate that deuterated secondary alcohol, whenadministered either topically or orally, results in readily detectabledeuterated VOCs (d6-acetone) in the exhaled breath for definitiveconfirmation of medication adherence, albeit with different kinetics ofappearance depending on the mode of delivery (oral or transdermal).

Example 6 (Ester Example 1)—GRAS Agent Listed as FoodAdditive—Aspartame: An Ester Food Additive Metabolized by Human GutEsterases and Gut Peptidases (See FIG. 43)

Drug Class: Food additive, considered GRAS by FDA; artificial sweetener

Mechanism: mimics the taste of sugar in humans Enzyme(s) for Metabolism:rapidly metabolized by human gut esterases and gut peptidases in humans

Metabolites: L-aspartic acid+L-Phenylalanine+Methanol

NICE Embodiment—Chemical Group Site(s) of Isotopic Label(s) on ParentMolecular Structure: Preferred site is the methyl group on Aspartame(indicated by red circle) but may include other locations on the parentmolecule.

NICE Embodiment—Type of Isotopic Labeling on Preferred Site(s): Insertisotopic label(s) on the preferred site, including but not limited to a)a single label of a given isotope type (e.g., one Deuterium label=CDH2)on the preferred site(s), b) multiple labels of a given isotope (e.g.,greater than one deuterium=CD2H or CD3) on the preferred site(s), or c)combinations of different types and numbers of isotopes (e.g.,deuterium, carbon and/or oxygen=13CDH2, 13CHD2, or 13CD3) on one or morelocations of the preferred site(s).

NICE Embodiment—Preferred Labeled Entity for Detection: isotopic (e.g.,deuterium) labeled methanol in the breath; a less preferred embodimentwould be labeled metabolic products of methanol (formaldehyde, formicacid and/or CO2—See FIG. 7 for details of metabolism of methanol).Isotopic labeling of larger metabolic fragments derived from the parent,which could be semi-volatile or non-volatile, could also serve asi-EDIMs.

Example 7 Esterase Example 2 FDA Approved Drug—Aspirin (AcetylsalicylicAcid): An Ester Drug Metabolized by Aspirin Esterases in Humans

(See FIG. 44)

Drug Class: Over the counter (OTC) drug

Mechanism: Nonsteroidal anti inflammatory drug (NSAID)—irreversiblyinhibits cyclooxygenase (COX) via acetylation of the serine residue atthe active site of COX, which suppresses production of prostaglandinsand thromboxanes

Enzyme(s) for Metabolism: Acetylsalicylic Acid (ASA) esterases

Metabolites: 2 acids (salicylic acid and acetic acid) NICEEmbodiment—Chemical Group Site(s) of Isotopic Label(s) on ParentMolecular Structure: Preferred site is the methyl group on ASA(indicated by red circle) but may include other locations on the parentmolecule.

NICE Embodiment—Type of Isotopic Labeling on Preferred Site(s): NICEEmbodiment—Type of Isotopic Labeling on Preferred Site(s): Insertisotopic label(s) on the preferred site, including but not limited to a)a single label of a given isotope type (e.g., one Deuterium label=CDH2)on the preferred site(s), b) multiple labels of a given isotope (e.g.,greater than one deuterium=CD2H or CD3) on the preferred site(s), or c)combinations of different types and numbers of isotopes (e.g.,deuterium, carbon and/or oxygen=13CDH2, 13CHD2, or 13CD3) on one or morelocations of the preferred site

NICE Embodiment—Preferred Labeled Entity for Detection: isotopic (e.g.,deuterium) labeled acetic acid in the breath; a less preferredembodiment would be labeled metabolic products of acetic acid, CO2.Isotopic labeling of larger metabolic fragments derived from the parent,which could be semi-volatile or non-volatile, could also serve asi-EDIMs, particularly if the liquid phase of breath is being analyzed.

Example 8 (Ester Example 3)—GRAS Agents Listed as Food Additives—Methyl,Ethyl, Propyl and Butyl Parabens: Ester Food Additives Metabolized byHuman Carboxylesterases and Tissue Esterases

(See Table 4)

Drug Class: Paraben′ is an abbreviation for para-hydroxybenzoic acid.Parabens are a family of alkyl esters of para-hydroxybenzoic acid thatdiffer at the para position of the benzene ring. There are four widelymarketed para-hydroxybenzoic acid (POHBA) esters: methylparaben,ethylparaben, propylparaben, and butylparaben. Used as foodadditives/preservatives; considered GRAS by FDA; Europe uses as ADI(acceptable daily intake) up to 10 mg/kg per day for methyl and ethylparaben

Mechanism: inhibits bacterial growth; food additive Enzyme(s) forMetabolism: rapidly metabolized by carboxylesterases and tissueesterases in humans Metabolites: para-hydroxybenzoic acid(POHBA)+corresponding alcohol (see below for details)

NICE Embodiment—Chemical Group Site(s) of Isotopic Label(s) on ParentMolecular Structure: Preferred site is the methyl group on Aspartame(indicated by red circle) but may include other locations on the parentmolecule.

NICE Embodiment—Type of Isotopic Labeling on Preferred Site(s): Insertisotopic label(s) on the preferred site, including but not limited to a)a single label of a given isotope type (e.g., one Deuterium label=CDH2)on the preferred site(s), b) multiple labels of a given isotope (e.g.,greater than one deuterium=CD2H or CD3) on the preferred site(s), or c)combinations of different types and numbers of isotopes (e.g.,deuterium, carbon and/or oxygen=13CDH2, 13CHD2, or 13CD3) on one or morelocations of the preferred site(s).

NICE Embodiment—Preferred Labeled Entity for Detection: isotopic (e.g.,deuterium) labeled alcohols in the breath; a less preferable embodimentis labeled distal metabolic products of the alcohols and acids generatedfrom the different parabens. Isotopic labeling of larger metabolicfragments derived from the parent, which could be semi-volatile ornon-volatile, could also serve as i-EDIMs, particularly if the liquidphase of breath is being analyzed.

TABLE 4 GRAS Agents Listed As Food Additives-Methyl, Ethyl, Propyl andButyl Parabens: Ester food additives metabolized by humancarboxyesterases and tissue esterases Molecular Breath Paraben StructureChemical Properties Metabolites Methyl paraben (Methyl-4-Hydroxybenzoate)

CAS: 99-76-3 MF: C8H8O3 MW: 152.15 MP: 126° C. SOLID Methanol + para-hydroxybenzoic acid (POHBA) Ethyl paraben Ethyl-4- Hydroxybenzoate

CAS: 120-47-8 MF: C₉H₁₀O₃ MW: 166.1766 BP: 297° C. MP: 117° C. SOLIDEthanol + POHBA Propyl paraben Propyl-4- Hydroxybenzoate

CAS: 94-13-3 MF: C₁₀H₁₂O₃ MW: 180.20348 MP: 97° C. SOLID Propanol +POHBA Butyl paraben Butyl-4- Hydroxybenzoate

CAS: 94-26-8 MF: C₁₁H₁₄O₃ MW: 194.23036 MP: 70° C. SOLID Butanol + POHBA

Example 9 (Esterase Example 4)—FDA Approved Drug—Clofibrate: An EsterDrug Metabolized by Esterases in Humans

(See FIG. 45)

Drug Class: Prescription

Mechanism: Hypolipidemic drug, known to induce peroxisome proliferation;a member of a large class of diverse exogenous and endogenous chemicalsknown as peroxisome proliferators; Activation of the peroxisomeproliferator activated receptor- (PPAR-α) key aspect of efficacyEnzyme(s) for Metabolism: Human Esterases

Metabolites: Carboxylic acid derivative of Clofibrate+Ethanol

NICE Embodiment—Chemical Group Site(s) of Isotopic Label(s) on ParentMolecular Structure: Preferred site is the ethyl group on clofibrate,particularly on the methyl group (indicated by red circle) but mayinclude other locations on the parent molecule.

NICE Embodiment—Type of Isotopic Labeling on Preferred Site(s): Insertisotopic label(s) on the preferred site, including but not limited to a)a single label of a given isotope type (e.g., one Deuteriumlabel=CH2CH2D) on the preferred site(s), b) multiple labels of a givenisotope (e.g., greater than one deuterium=CH2CHD2, CH2D3, CHDCD3,CD2CD3) on the preferred site(s), or c) combinations of different typesand numbers of isotopes (e.g., deuterium, carbon and/or oxygen on one ormore locations of the preferred site(s).

NICE Embodiment—Preferred Labeled Entity for Detection: isotopic (e.g.,deuterium-based) labeled ethanol in the breath; a less preferredembodiment would be labeled metabolic products of ethanol. Isotopiclabeling of larger metabolic fragments derived from the parent, whichcould be semi-volatile or non-volatile, could also serve as i-EDIMs,particularly if the liquid phase of breath is being analyzed.

Example 10 (Esterase Example 5)—FDA Approved Drug—Esmolol: A DrugMetabolized by Arylesterase Located within the Cytosol of Human RedBlood Cells

(See FIG. 46)

Drug Class: Controlled/prescription drug Mechanism: Ester-based ultrashort acting beta blocker that is beta1 receptor selective

Enzyme(s) for Metabolism: In contrast to most ester-containing drugs,the hydrolysis of esmolol is mediated by an esterase in the cytosol ofred blood cells (RBC) called arylesterase.

Metabolites: carboxylic acid derivative of Esmolol+Methanol

NICE Embodiment—Chemical Group Site(s) of Isotopic Label(s) on ParentMolecular Structure: Preferred site is the methyl group on esmolol(indicated by red circle) but may include other locations on the parentmolecule.

NICE Embodiment—Type of Isotopic Labeling on Preferred Site(s): NICEEmbodiment—Type of Isotopic Labeling on Preferred Site(s): Insertisotopic label(s) on the preferred site, including but not limited to a)a single label of a given isotope type (e.g., one Deuterium label=CDH2)on the preferred site(s), b) multiple labels of a given isotope (e.g.,greater than one deuterium=CD2H or CD3) on the preferred site(s), or c)combinations of different types and numbers of isotopes (e.g.,deuterium, carbon and/or oxygen=13CDH2, 13CHD2, or 13CD3) on one or morelocations of the preferred site(s).

NICE Embodiment—Preferred Labeled Entity for Detection: isotopic (e.g.,deuterium) labeled methanol in the breath; a less preferred embodimentwould be labeled metabolic products of methanol. Isotopic labeling oflarger metabolic fragments derived from the parent, which could besemi-volatile or non-volatile, could also serve as i-EDIMs, particularlyif the liquid phase of breath is being analyzed.

Example 11 (CYP450 Example 1)—CYP-3A4-Mediated Metabolism FDA ApprovedDrug: Verapamil—an L-Type Calcium Channel Blocker

(See FIG. 47)

Verapamil(2,8-bis-(3,4-dimethoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile) isa L-type calcium channel blocker that liberates formaldehyde uponoxidative dealkylation (N-demethylation) by CYP-3A4. Orally administeredverapamil undergoes extensive metabolism in the liver. One majormetabolic pathway is the formation of norverapamil (N-methylatedmetabolite of verapamil) and formaldehyde by CYP-3A4. Although dependentupon the number of alternate metabolic pathways, the rate of formationof a specific metabolite(s) (i.e., verapamil->norverapamil andformaldehyde via CYP-3A4) generally appears to be predictive of in vivofunctional enzyme competence. In fact verapamil is metabolized byO-demethylation (25%) and Ndealkylation (40%). The CYP-3A4 is most theimportant enzyme in humans for metabolizing drugs. It has been estimatedthat the CYP-3A4 isoform of the P450 system is responsible formetabolizing 55-60% of all pharmaceutical agents. The CYP3A4 plays acritical role in metabolizing many drugs, including several cytotoxicdrugs such as paclitaxel, docetaxel, vinorelbine, vincristine,irinotecan, topotecan, ifosfamide, cyclophosphamide, and tamoxifen.Thus, alterations in CYP-3A4 function frequently lead to drug-inducedincreases in morbidity and mortality. The isotopic labels shown in Table2 (preferably deuterium), where appropriate, can be used to labelvarious atoms (red circle) of verapamil, which in turn, will generateisotopic-labeled formaldehyde that will serve as the preferredembodiment of the i-EDIM in this example. In addition, isotopic labelingof larger metabolic fragments (e.g., norverapamil, etc.) derived fromthe parent, which could be semi-volatile or nonvolatile, could alsoserve as i-EDIMs, particularly if the liquid phase of breath is beinganalyzed.

Example 12 CYP450 Example 2—CYP-3A4-Mediated Metabolism FDA ApprovedDrug: Amiodarone—an Antiarrhythmic Drug

(See FIG. 48)

Amiodarone is one the most effective antiarrhythmic drugs in clinicalmedicine. It is highly effective in treating atrial fibrillation,particularly in preventing its re-occurrence. Although this drug has acomplex mechanistic profile (blocks sodium channels, beta receptors,calcium channels, and potassium channels) its major electrophysiologicalaction is to prolong repolarization in cardiac tissue, predominantly byblocking potassium channels. Therefore, it is classified as a Class IIIantiarrythmic drug according to the Vaughn-William Classification. Theisotopic labels shown in Table 2 (preferably deuterium), whereappropriate, can be used to label various atoms (red circle) ofamiodarone, which in turn, will generate isotopic-labeled acetaldehydethat will serve as the preferred embodiment of the i-EDIM in thisexample. In addition, isotopic labeling of larger metabolic fragmentsderived from the parent, which could be semi-volatile or nonvolatile,could also serve as i-EDIMs, particularly if the liquid phase of breathis being analyzed.

Example 13 CYP450 Example 3—CYP-3A4-mediated Metabolism FDA ApprovedDrug: Propafenone—An Antiarrhythmic Drug

(See FIG. 49)

Propafenone is an antiarrhythmic drug that acts by primarily blockingsodium channels, and is classified as a Class IC antiarrythmic drugaccording to the Vaughn-William Classification. The isotopic labelsshown in Table 2 (preferably deuterium), where appropriate, can be usedto label various atoms (red circle) of propafenone, which in turn, willgenerate isotopic-labeled propionaldehyde that will serve as thepreferred embodiment of the i-EDIM in this example. In addition,isotopic labeling of larger metabolic fragments derived from the parent,which could be semivolatile or non-volatile, could also serve asi-EDIMs, particularly if the liquid phase of breath is being analyzed.

Example 14 CYP450 Example 4—CYP-3A4-mediated Metabolism FDA ApprovedDrug: Diltiazem—An Antiarrhythmic Drug

(See FIG. 50)

Diltiazem is a L-type calcium channel blocker, which undergoes complexbiotransformation, including deacetylation, N-demethylation, andOdemethylation. Of these pathways, CYP-3A4 probably plays a moreprominent role than CYP2D6 in the metabolism of diltiazem. The isotopiclabels shown in Table 2 (preferably deuterium), where appropriate, canbe used to label various atoms (red circle) of diltiazem, which in turn,will generate isotopic-labeled formaldehyde and/or acetic acid that willserve as the preferred embodiments of the i-EDIMs in this example. Inaddition, isotopic labeling of larger metabolic fragments derived fromthe parent, which could be semi-volatile or non-volatile, could alsoserve as i-EDIMs, particularly if the liquid phase of breath is beinganalyzed.

Example 15 CYP450 Example 5—CYP-2D6-Mediated Metabolism FDA ApprovedDrug: Codeine—a Prodrug Narcotic for Analgesia

(See FIG. 51)

Shown is an example where the CYP substrate is a prodrug (codeine) thatis converted by the P450 system (CYP 2D6) into the active drug(morphine). Morphine has a significantly higher affinity for the μopioid receptor than codeine, and thus is thought to mediate theanalgesic properties of codeine. Only about 10% of codeine is normallyconverted to morphine in vivo. In this embodiment, the NICE system couldbe used to not only ensure that codeine is efficacious (i.e., ensuresadequate conversion to morphine) but also to ensure that an inordinateamount of codeine isn't converted to morphine if a subject has a superfunctional of CYP-2D6. The latter scenario would cause an adverse drugreaction (ADR) because an excessive amount of morphine would be presentin the body. Likewise, in the former scenario, the NICE system wouldidentify those subjects that wouldn't get adequate pain relief from thisdrug, because not enough morphine is produced from codeine. The functionof CYP 2D6 is altered by a great many factors including but not limitedto genetics or drug-drug interactions. For example, because 6-10% ofCaucasians have poorly functional CYP2D6, they do not get adequate painrelief from codeine. Furthermore, a number of medications are potentCYP2D6 inhibitors and reduce or even completely eliminate the efficacyof codeine. The most notorious of these are the SSRIs includingfluoxetine (Prozac) and citalopram (Celexa). The high end PO dose ofcodeine is typically 240 mg given over 24 hours. The small arrowindicates the site of catalytic action by the CYP enzyme to liberate theformaldehyde. The isotopic labels shown in Table 2 (preferablydeuterium), where appropriate, can be used to label various atoms (redcircle) of codeine, which in turn, will generate isotopic-labeledformaldehyde that will serve as the preferred embodiment of the i-EDIMin this example. In addition, isotopic labeling of larger metabolicfragments derived from the parent, which could be semi-volatile ornon-volatile, could also serve as i-EDIMs, particularly if the liquidphase of breath is being analyzed.

Example 16 CYP450 Example 6—CYP-1Δ2-Mediated Metabolism FDA ApprovedDrug: Olanzapine—an Antipsychotic Agent

(See FIG. 52)

Olanzapine is one of the most widely used antipsychotic drugs in theworld. It is used to treat schizophrenia. The major metabolic pathwayfor olanzapine is mediated by CYP-1Δ2. Its metabolism is well predictedby using the caffeine breath test as a probe to examine the ability ofthe CYP450 system to metabolism olanzapine. The small arrow indicatesthe site of catalytic action by the CYP enzyme to liberate theformaldehyde. The isotopic labels shown in Table 2 (preferablydeuterium), where appropriate, can be used to label various atoms (redcircle) of olanzapine, which in turn, will generate isotopic-labeledformaldehyde that will serve as the preferred embodiment of the i-EDIMin this example. In addition, isotopic labeling of larger metabolicfragments derived from the parent, which could be semivolatile ornon-volatile, could also serve as i-EDIMs, particularly if the liquidphase of breath is being analyzed.

Example 17 CYP450 Example 7—CYP-1Δ2-mediated Metabolism Class 1 Drug:Caffeine—A Food Additive

(FIG. 53)

Caffeine is a xanthine-type drug that is widely found in many foods,including beverages. Caffeine is a central nervous stimulant. It hasbeen generally accepted as a specific in vivo probe for CYP1Δ2 activity.Approximately 80% of caffeine given orally to humans is converted totheophylline. Caffeine has been shown to provide an accurate phenotypicprobe for measuring CYP1Δ2 activity, particularly when predicting theability of olanzapine to be metabolized in vivo. The small arrowindicates the site of catalytic action by the CYP enzyme to liberate theformaldehyde. The isotopic labels shown in Table 2 (preferablydeuterium), where appropriate, can be used to label various atoms (redcircle) of caffeine, which in turn, will generate isotopic-labeledformaldehyde that will serve as the preferred embodiment of the i-EDIMin this example. In addition, isotopic labeling of larger metabolicfragments derived from the parent, which could be semi-volatile ornon-volatile, could also serve as i-EDIMs, particularly if the liquidphase of breath is being analyzed.

Example 18 Approaches to Assessing Medication Adherence Using “Cold”Isotopic (Deuterium)-Based Chemistry and C—H (C-D) StretchingVibrational Modes in the Mid-IR Region

Using a ThermoFisher Nicolet 6700 FT-IR Spectrometer with 16-L GeminiLong Path Gas Cell, FTIR Conditions: Auxiliary Experiment Module: HighResolution Gas Sampling with MCT/A Detector (cooled with liquidnitrogen), KBr Beam Splitter. Range: 4000-650 cm⁻¹, Gain: 8, Aperture:4.

Fill two 5-L Tedlar gas sampling bag with blank breath. Allow each bagto sit for at least 1 hour. Add 1 μL of neat volatile organic compound(e.g., acetone, d6-acetone, isopropanol) to one of the 5-L Tedlar gassampling bags filled with blank breath. Allow this bag to sit for atleast 1 hour. Evacuate the gas cell then fill the gas cell with thecontents of the 5-L Tedlar gas sampling bag filled with blank breath.Collect a background spectrum. Evacuate the gas cell then fill the gascell with the contents of the 5-L Tedlar gas sampling bag containing thevolatile organic compound in blank breath. Collect a sample spectrum.See FIGS. 23-39 for results.

Example 19 Detection of Breath Acetone Using Mass Spectroscopy andmGC-MOS: Isopropanol and Perdeuterated Isopropanol as Adherence EnablingMarkers (i-AEMs) in the SMART™ Adherence System

Isopropanol (IPA) and acetone are listed by the FDA as direct foodadditives (GRAS). Isopropanol and acetone are listed as excipients inthe FDA's IIG list. Isopropanol and acetone are listed in the FDA's Q3Cguidance. Class III Solvent: 50 mg or less, no concern. PermissibleDaily Exposure (PDE): IPA 138 mg/day orally; and acetone 210 mg/dayorally; Deuterations deemed safe by FDA.

Note: In humans acetone has a metabolic t_(1/2)=17-27 hrs, see, forexample, Jones-A W, J Analytical Toxicology, 24:8-10, 2000.

See FIG. 55, which provides a breath profile (exhalation provided toOrbiTrap, LC/MS/MS) from a 30 mg bolus of IPA delivered in a size 0capsule to a fasting subject, showing IPA induced increase abovebaseline for acetone in the exhaled breath of the subject. See FIG. 55for mGC analysis after ingestion of 10 mg IPA. FIG. 55 A shows the firstderivative of the mGC profile for 0, 5, 10, 15, and 30 minutes postingestion of 10 mg IPA. These results were obtained even withoutoptimization of the mGC for “early eluters” (system peak, isoprene andacetone). FIG. 55B shows the ratio of first derivatives for theacetone/isoprene mGC profiles.

From these studies, we conclude that low quantities of isopropanol areeffective to serve as an AEM but even more so as an i-AEM using the mGC,(i.e. with or without the need for deuterations to document adherence.Isopropanol could generate either a primary (acetone alone) or secondarybreath marker (e.g., acetone+2-butanone) to document adherence. Giventhe long half-life of acetone in humans (17-27 hrs), use of this breathmarker could serve as a marker of chronic adherence, and couldcomplement the “acute” adherence measurement made using the breathmarker, 2-butanone.

Note: In humans acetone has been reported to have a metabolict_(1/2)=17-27 hrs, see, for example, Jones-A W, J Analytical Toxicology,24:8-10, 2000.

Example 20 GC/MS and OrbiTrap (LC/MS/MS) Analysis

Protocol: Participant ingested 100 mg of d8-2 propanol in a size 3 LiCap2 h after lunch. Breath samples were analyzed by LC/MS and GC/MS for thepresence of d8-Acetone. GC/MS samples were collected at 0, 5, and 15 minafter ingestion. Direct breath samples (4 s per breath) were analyzed byLC/MS for 270 minutes after ingestion of the pill using the ESI sourcewith ammonia modified water (0.1%) as an ionizing solution.

Note: Acetone (m/z 58, [(CH3)2C═O)]+); d8-Acetone (m/z 64,[(CD3)2C=0)]+).

See FIGS. 56-59.

Example 21 Real Time Analysis of Acetone Breath Kinetics FollowingIngestion of 3 mg d8-Isopropanol Using the OrbiTrap LC/MS/MS

See FIGS. 60-61.

ESI Direct exhaled breath analysis after ingestion of 3 mgd8-isopropanol in a size 3 LiCap. Selected ion chromatogramscharacteristic for NH4 Adducts of Acetone and d6-Acetone.

Ingestion Capsule was ingested immediately after subject consumed acarbohydrate meal. In FIG. 61C, two y axis scales are included to showdifferences, with the left axis=endogenous acetone, and the rightaxis=d6-Acetone.

Example 22 Real Time Analysis of Acetone Breath Kinetics FollowingIngestion of 10 mg d8-Isopropanol and 10 mg Isopropanol Using theOrbiTrap LC/MS/MS

See FIG. 62. Breath kinetics of acetone and d6-acetone following thesimultaneous ingestion of 10 mg 2-propanol and 10 mg d8-2-propanol in asize 3 LiCap. The subject ingested one capsule containing the 20 mgmixture at t=0 and a second capsule ˜50 min later. A rise in bothacetone and the deuterated analog is apparent within 2 min followingadministration of either dose. Proportional rise in peak height indicatedeuterations do not have a significant effect on the metabolicconversion to acetone from IPA by secondary alcohol dehydrogenase (2°ADH).

Example 23 Breath Kinetics of Exhaled 2-Butanol and 2-Butanone Followingthe Concurrent Ingestion of 2-Butanol and Ethanol

Instrumentation and Methods:

20 mg of 2-butanol in a size 3 LiCap was ingested along with one shot(˜44 mL) of 100 proof ethanol (50% v/v). Following ingestion, ethanol,acetone, 2-butanol and 2-butanone levels were monitored in exhaledbreath samples using the LTQ-LCMS. Single 5 s breath samples wereadministered directly into the modified ESI source at 5 min intervalsfor 45 min.

The ESI source was operated in positive ion mode. A 0.2% NH₄OH:watermobile phase was introduced into the source at a flow rate of 0.1 mL/minduring sampling to produce ammonium adducts of the analytes of interest.

FIG. 63: A. Mass spectrum of a single breath sample taken before theingestion of 2-butanol with ethanol. Of the four analytes highlighted,only acetone can be positively identified. The small peak at 90 islikely due to isotopic interference from the unknown backgroundcomponent appearing at m/z=88 and not 2-butanone; B. Mass spectrum of asingle breath sample taken 5 min after the ingestion of 2-butanol andethanol. Ethanol, 2-butanone and acetone are now present as prominentpeaks, but 2-butanol is barely detectable above baseline.

FIG. 63 shows the peak height of each ion of interest as a function oftime to yield the breath kinetics for each breath marker. Even with areasonable dose of ethanol present in the stomach, the kinetics of2-butanone appears unaffected (or at least very similar to a typicalresponse following the ingestion of just 2-butanol) and no significant2-butanol was detected; C. Breath kinetics of 2-butanone and d6-acetonefollowing ingestion of neat 2-butanol (40 mg) and d8-isopropanol (20 mg)after lunch, baseline breath; D. 5 minutes post ingestion; E. 25 minutespost ingestion; F. D6-Acetone was detectable in the breath one minuteafter ingestion of the d8-isopropanol. The graph was generated usingdata from orbitrap LCMS. The orbitrap was configured to capturesequential spectra (˜5 spectra per second) and these spectra wererecorded for the duration of the experiment (60-90 min usually) toproduce a real time continuous trace. The electrospray interface on theorbitrap was modified to allow a subject to blow exhaled breath samplesdirectly into the source while the mass spectra were being collected.The rapid clearance of the breath samples from the source allowed us tocapture and characterize mass spectra from exhaled breath samples inreal time. In theory we could use the orbitrap to capture anddistinguish every exhaled breath that a subject makes during anexperiment but in practice we typically don't need to collect more thanone breath sample per minute.

FIG. 64 (A) Plotting the peak height of each ion of interest as afunction of time yields the breath kinetics for each potential breathmarker. Even with a reasonable dose of ethanol present in the stomach,the kinetics of 2-butanone appears unaffected (or at least very similarto a typical response following the ingestion of just 2-butanol) and nosignificant 2-butanol was detected; (B) Breath kinetics of 2-butanoneand d6-acetone following ingestion of neat 2-butanol (40 mg) andd8-isopropanol after lunch.

Example 24 FTIR Analysis of Actone and Isopropyl Alcohol Along withtheir Perdeuterated Isotopologues

Comparison of NIST Webbook Gas Phase IR Spectra for Isopropyl Alcoholversus one obtained using the UF Nanomedicine Thermo Nicolet 6700 FTIRis shown in FIG. 65. In FIG. 65A, there is provides a tracing showingthe infrared spectrum from a NIST Webbook Gas Phase IR Spectrum of2-Propanol (seehttp://webbook.nist.gov/cgi/cbook.cgi?ID=C67630&Units=SI&Type=IR-SPEC&Index=2#IR-SPEC) whereas in FIG. 65B, there is provided aspectrum obtained by the inventors using a Thermo Nicolet 6700 FTIR GasPhase IR Spectrum of 2-Propanol. The reproducibility of the spectra areclear.

In FIG. 66A there is provided a tracing of the FTIR analysis of acetoneand d6-acetone showing clear areas where these spectra aredistinguishable from each other. FIG. 66A′ shows a detail of the regionbetween 3300 cm-1 to 2000 cm-1.

In FIG. 66B, there is provided a tracing of the FTIR analysis of IPA andd8-IPA, again showing clear areas where these spectra aredistinguishable from each other.

In FIG. 67, there is provided FTIR Spectra of Acetone and IsopropylAlcohol with their perdeuterated isotopologues, with a detail of eachtracing in the Fingerprint Region (1170 cm⁻¹ to 1300 cm⁻¹, 8.5470 mm to7.6923 mm). The ability of this technique to distinguish between theperdeuterated and non-deuterated molecules is clear. The lines drawn at1252.56 cm⁻¹=7.9836 μm and 1228.21 cm⁻¹=8.1419 μm indicate optimalwavenumbers to monitor acetone and deuterated acetone in a smallwavenumber window (24 cm-1).

In FIG. 68 there is provided, in FIG. 68A, FTIR Spectra of d6-acetoneversus Blank Breath, with details of portions of these spectra beingshown in FIGS. 68B and 68C. As can be seen, there are clear portions ofthese spectra which are not interfered with by compounds in theendogenous breath, making it clear that the d6-acetone is an excellenti-EBM.

Example 25 Use of an i-API as its Own i-AEM to Produce a Specific andCognate i-EBM

Those skilled in the art will appreciate from the present disclosurethat in one preferred mode of practicing this invention, the i-AEM is amarker that is included in a dosage form for delivery to a subject atthe same time that an API is delivered to a subject, to enableconfirmation (by detection of the i-EBM in the breath produced from thei-AEM) of delivery of the API to the subject. In another preferred modeof carrying out the present invention, however, the API itself mayinclude a non-ordinary but stable isotope and thereby can act as its owni-AEM, to produce in the exhaled breath, an i-EBM specific to that API.One example of such a system involves the use of a deuterated form ofpropofol. Propofol is detected in the exhaled breath after intravenousadministration of propofol to the subject (see U.S. Pat. Nos. 6,981,947,and 7,104,963 and their related foreign counterparts). In a particularapplication, where it is advantageous to measure an i-EBM in the exhaledbreath, inclusion of a fraction of deuterated propofol (i-propofol) inthe propofol that is administered intravenously, permits detection ofthe i-propofol or metabolites thereof in the exhaled breath can providedata that might not otherwise be available. Of course, many other i-APIsmay be contemplated for use according to this invention with anappropriate SMART® device. In fact, Concert Pharmaceuticals, Inc.,(Lexington, Mass.), has announced that it “uses deuterium-basedchemistry to create and develop highly differentiated new medicines byleveraging decades of pharmaceutical and clinical experience to reducetime, risk and expense.” Such compounds would serve as i-AEMs forthemselves, either as the cognate parent compounds or as metabolitesthereof, which appear as i-EBMs in the exhaled breath. Of course, incombination with the i-API, additional i-AEMs may be included to assistrefinement of the i-EBM analysis—such that different half lives ofdifferent species may be determined. As a result, quite specificadherence data is made possible at a much more refined degree ofgranularity than has ever before been available. Thus, for example,i-EBMs generated from the i-API may have a half life of several minutes,while i-EBMs generated from exogenous i-AEMs (i.e. i-AEMs that are notthe i-API itself or any part of the i-API, but included with the i-APIin a dosage form), may exhibit half lives of several hours to days. Bymeasuring both types of i-EBMs in the exhaled breath utilizing theSMART® technology, it is possible to determine total dosage adherenceinformation, including when a given dose was taken, which dose/doseswere missed and when, and the like. Thus, for example, i-IPA (e.g.,deuterated isopropyl alcohol) produces i-acetone (e.g., deuteratedacetone) which can be detected in the exhaled breath for several days,while i-2-butanol (e.g., deuterated 2-butanol) gives rise to i-butanone(e.g., deuterated butanone) which exhibits a very short half life ofseveral minutes to one or two hours in the exhaled breath.

Example 26 Breath Kinetics of Exhaled d6-Acetone Following the Ingestionof 100 mg of d8-Isopropanol Per Diem for 5 Days

100 mg aliquots (125 μL) of neat d8-isopropanol were ingested at 24 hintervals for a period of 5 days. Each aliquot was administered in asize 0 LiCap along with ˜200 mL of water.

Throughout the study, acetone and d6-acetone levels were monitored inexhaled breath using the LTQ-LCMS. Each breath sample consisted of asingle full breath exhaled directly into the modified ESI source. TheESI source was operated in positive ion mode using a 0.1% NH₄OH:watermobile phase (at 0.1 mL/min) as an ionizing medium. Both acetone andd6-acetone were present as protonated ammonium adducts and monitoredusing m/z=76 and m/z=82, respectively.

Following the ingestion of each aliquot, breath samples were taken every2 min for the first 30 min following ingestion, every 5 min from 30-60min after ingestion and every 10 min from 60-120 min. Additional breathsamples were collected at ˜9 h and 20 h after ingestion. After the finaldose, breath samples were taken once every 24 h for the next 3 days(total study time=8 days).

FIG. 70 shows that native acetone peak heights remained reasonablyconstant throughout the study.

FIG. 71 shows that baseline levels for ion 82 (the ion used to monitord6-acetone) were low and less than 1000 (<1% of typical acetone levels).An increase of exhaled d6-acetone was apparent within 2-4 minutes ofingesting each dose of d8-IPA. Maximum breath levels were achieved after1-2 h and ranged from 450,000 to 800,000 peak height. (˜2-5× nativeacetone).

FIG. 72 shows that 24-hour trough levels were relatively unchanged overthe course of the study and were ˜10% of peak maximum:

Day Trough Peak Height 1 51988 2 54470 3 47369 4 54955 5 62324

FIG. 73 shows that the decline of d6-acetone in exhaled breath followeda first order decay (2-24 h post ingestion). The rate constant (k) forthis decay was consistent throughout the study:

Day k (h⁻¹) 2 −0.1045 3 −0.1123 4 −0.1034 5 −0.1113 Average k = −0.1079t½ ≃ 6.4 h

FIG. 74 shows that at this rate of elimination, approximately 6-10% ofmaximum peak response remains after 24 h. Such kinetics should producesteady-state trough levels that are also ˜10% of the maximum peak. Thismatches the observed trough levels during the study. 21 h after thefinal dose, exhaled d6-acetone produced a peak height of 62000, which is34% of the average acetone level measured during the study. By 45 h, thed6-acetone level had fallen to 3886. d6-Acetone returns to baseline(more specifically, ion 82 levels return to baseline) after −65 hours.

Example 27 Isopropyl Alcohol (IPA) as an AEM Using a SMART® Type IDevice

An mGC-MOS device was used to analyze breath samples before and afteringestion of 100 mg of Isopropyl Alcohol (IPA). Standards at fourdifferent concentrations (333, 666, 1662, and 3323 ppb-moles) werecreated by adding neat quantities of IPA to UHP nitrogen in 1-L Tedlargas sampling bags (SKC Inc, Eighty Four, Pa.). The mGC-MOS chromatogramsare shown in FIG. 77a , and the calibration curve for these standards isshown in FIG. 77b For the first 5 days of this study, a placebo capsulecontaining 100 mg of water was ingested each morning and breath sampleswere analyzed using a mGC-MOS device at 0, 15, and 30 minutes. For thelast 5 days of this study, a capsule containing 100 mg of isopropylalcohol was ingested each morning and breath samples were analyzed atthe same time points as in the placebo part of this study (0, 15, and 30minutes). The results from this study are shown in FIG. 77c . Note howthe ingestion of IPA (100 mg) rapidly (15 and 30 min after oralingestion of IPA) increased the breath acetone levels by greater than 6×those of baseline acetone concentrations. This rise in breath acetoneconcentration is very distinctive in terms of documenting medicationadherence, because the human body when resting in the home settingcarrying out adherence measurements could not generate this type of risein endogenous acetone levels over this short (15 and 30 min) a timeframe. Consistent with a half life of less than 10 hours in breath inthis subject, the trough concentrations of acetone rapidly reachedsteady state within 1-2 days, and are consistent with those obtained inanother subject, who ingested 100 mg d8-IPA and used breath analysis bythe OrbiTrap (FIG. 71).

Example 28 AMAM, IMAM and CMAM Using the Smart System According to thisInvention

In implementing a SMART® system according to this invention for use as a“gold standard” for acute medication adherence monitoring (AMAM;adherence assessment “look back window” up to 2-3 hours), intermediatemedication adherence monitoring (IMAM; adherence assessment “look backwindow” up to 24 hours), and/or chronic medication adherence monitoring(CMAM; adherence assessment “look back window” that is three days toeven weeks), five key inter-related factors are involved:

Factor 1: the half life of the EDIM in humans;

Factor 2: the concentration of EDIM in breath;

Factor 3: the limit of detection (LoD) of the sensor to detect the EDIMin breath;

Factor 4: the level of any background EDIM or interferents that canmimic the EDIM on the sensor, which may be present in breath; and

Factor 5: significant absorption of the AEM from the stomach (e.g.,adequate AEM permeability through the thick gastric epithelium), whichis a requirement for AMAM capabilities but not for CMAM or IMAMcapabilities.

By orchestrating the quartet of factors (1-4 above, i.e. byadministering a dose of AEM to generate the highest concentration ofEDIM having the longest half life in breath, which in turn is detectedwith the most sensitive sensor that has no background interference inbreath, including no EBM already present in breath (e.g., no endogenousacetone) or no other breath markers that could mimic the presence of theEBM to the sensor), a SMART® architecture for a CMAM system with thelongest (days to weeks) “look back” time window in terms of assessingadherence is enabled. As shown in multiple examples herein, thisframework is preferably (but not exclusively) achieved by using coldisotopologues of AEMs that generate highly distinctive coldisotopologue-based EDIMs, which are detected by Type 1 or, preferably,Type (e.g. infrared-based) SMART® devices according to this invention.This approach also makes it feasible to use very low quantities of AEM,including the formulation strategy of simply spraying a few mg of coldisotopologue-based AEM(s) on the surface of a solid oral dosage form(SODF) (and, depending on volatility, overcoating to entrap and preventloss of the AEM). In contrast, the quartet of factors consisting of adose of AEM that generates the lowest concentration of an EDIM havingthe shortest half life in breath, which in turn is detected with theleast sensitive sensor that has the most significant backgroundinterference (EBM already present in breath and/or the presence of otherbreath markers that can mimic the EBM to the sensor) provides for theshortest medication adherence assessment “look back” window period.Ideally, the optimal SMART® architecture for an AMAM system thatprovides a shorter (up to ≈1-2 hrs) adherence “look back” window periodentails production of an EDIM having a short half life in breath that isdetected with a sensor that is sensitive to the EDIM and has nobackground interference to contend with. Unlike a system designed onlyfor CMAM, one that encompasses AMAM with or without IMAM and/or CMAMcapabilities requires the AEM to have significant absorption in thestomach, so the EDIM is able to promptly appear in the breath viametabolism of the AEM, where a prompt rise above baseline breath EDIMlevels indicates acute adherence. If the AEM is absorbed in only thesmall intestine (e.g., duodenum), the appearance of the EDIM in breathis totally dependent on the highly variable periods of time it takes forthe stomach to empty its contents (e.g., AEM) into the upper smallintestine for absorption. In contrast, it is not a prerequisite withIMAM (up to 1 day adherence “look back” window) and CMAM (days to weeksadherence “look back” window) that the AEM have significant absorptionin the stomach to function properly, because duodenal absorption isadequate, given the longer half life of the EDIM used in CMAM. However,it is important to note that even an EDIM highly suitable for CMAM(longer half life in breath) can be effectively used for AMAM (pill bypill adherence assessments), if it is significantly absorbed from thestomach, and a baseline breath sample is obtained in addition to one ata slightly later time (e.g., 20-30 min) after ingestion of the AEM,because even compounds at steady state levels in the blood with longerhalf lives show a significant EDIM concentration rise with each dose.This rise above baseline (trough) levels of the EDIM is easily detectedwith a two breath script.

When humans orally ingest 2° alcohols as the AEM, the following findingshave been noted: 1) absorption of the 2° alcohols (AEMs) in thegastrointestinal tract is complete (fractional absorption is unity) andvery rapid, relative to the rate of metabolism of the AEM (2° alcohol)to the EDIM (ketone), 2) metabolism of the 2° alcohols to theircorresponding ketones is complete, given the high degree of 1^(st) passmetabolism, and 3) the concentrations of EDIM in breath rapidlyequilibrate with those in blood, and reflect the free concentration ofEDIM in blood (and plasma).

Considering these five factors as each relates to SMART® AdherenceSystem function:

Factor 1: EDIM Half Life in Breath

After ingesting various doses of AEMs (e.g., 2-butanol, isopropylalcohol), we have determined the EDIM breath concentration-timerelationships, including the half life in breath of various EDIMs,including 2-butanone (Example 3 and Morey et al, AIDS Behav17(1):298-306. 2013), 2-pentanone (Example 3), and acetone (Example 5;FIG. 74). As it relates to the SMART® Adherence System, theconcentration rise (rapid absorption of AEM and conversion to EDIM) anddecay of the EDIM with time in breath following oral administration ofthe AEM is well described by a 1 compartmental (monoexponential)pharmacokinetic (PK) model, reflecting absorption and elimination, whichcan be described by the following equation (dertived from Miller'sAnesthesia, 6th Edition, p 81, 2005, Ed: Ronald D. Miller, ElsevierChurchill Livingstone, Philadelphia, Pa.):

$\begin{matrix}{{C_{EDIM}(t)} = {C_{EDIMo}*F*\frac{k_{a}}{k_{a} - k_{e}}*\left( {^{{- k_{e}}t} - ^{{- k_{a}}t}} \right)}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

where:

C_(EDIM)(t)=concentration of EDIM in breath as a function of time (t);

C_(EDIMo)=maximum concentration of EDIM in breath derived from dividingthe dose of EDIM (complete conversion of AEM dose to EDIM) by the volumeof distribution (V_(d)) for the EDIM;

F=fraction bioavailable of AEM (complete conversion to EDIM, so F=1);

k_(a)=1^(st) order rate constant for absorption of AEM into thecompartment (absorption is very rapid for 2° alcohol AEM with completeconversion to corresponding ketone);

k_(e)=1^(st) order rate constant for elimination of the EDIM from thecompartment;

e=Euler's number, (2.71828 . . . ; i.e. the base for the naturallogarithm, whereby 1ne^(x)=x).

The time to attain the maximum concentration of EDIM in breath (T_(MAX))is given by the following equation:

$\begin{matrix}{T_{{MA}\; X} = {\frac{1}{\left( {{ka} - {ke}} \right)}*{\ln \left( \frac{ka}{ke} \right)}}} & {{Equation}\mspace{14mu} 2}\end{matrix}$

Following the oral ingestion of 2° alcohols (and most suitable AEMs),gastrointestinal absorption is complete and metabolic conversion to itscorresponding ketone is very rapid. Therefore, because k_(a)>>k_(e) andF is 1, Equation 1 simplifies to Equation 3:

C _(EDIM)(t)=C _(EDIMo) *e ^(−k) ^(e) ^(t)   Equation 3

The 1^(st) order rate elimination constant (k_(e)) of the EDIM isrelated to the elimination half life (t_(1/2a)) of the EDIM (timerequired for the EDIM concentration to fall by half in breath) by thefollowing equation:

$\begin{matrix}{t_{{1/2}e} = \frac{0.693}{ke}} & {{Equation}\mspace{14mu} 4}\end{matrix}$

Because the conversion of the AEM to the EDIM is complete and relativelyrapid, the t_(1/2e) provides an excellent measure of the time it takesto achieve steady state EDIM levels in breath, both trough (C_(Trough))and maximal (C_(MAX)), as it reflects blood levels of EDIM, after aconstant oral dosing regimen of the AEM is initiated (see FIG. 71 & FIG.77). Moreover, during dosing with AEMs, a time of four half lives isrequired to reach approximately 94% of steady state EDIM concentrationsin breath (see FIG. 78). The time to steady state is independent of thedose, but the fluctuations (concentration swing between each dosing) isproportional to the AEM dosage interval (T) divided by the EDIMelimination half life (t_(1/2a)) in breath. In addition, the steadystate concentration achieved with chronic oral AEM dosing isproportional to the AEM dose divided by dosage interval (T).

FIG. 78 provides a graphic representation of the fundamentalpharmacokinetic relationships for six successive administrations of anoral drug. The light line is the pattern of drug accumulation duringrepeated administration of a drug at an interval equal to itselimination half life, when drug absorption is very rapid relative toelimination. The concentration maxima approach 2 and the minima approach1 during the steady state. The heavy line depicts the pattern duringadministrating of equivalent dosage by continuous intravenous infusion.Curves are based upon a one compartment model. The x axis representstime, as indicated by multiples of elimination half life (t_(1/2a)).(Reference: modification of FIGS. 1-6, page 27, Goodman and Gilman, ThePharmacological Basis of Therapeutics, 8^(th) Edition, 1993, PergamonPress, New York, N.Y. Abbreviation Key: C_(Trough), trough concentrationof EDIM (circle symbols); C_(MAX), maximum concentration of EDIM inbreath (horizontal dotted lines).

As shown in FIG. 78, repeated oral administrations of the AEM producesaccumulation of the EDIM, the magnitude of which depends upon itselimination half life (t) and dosage interval (T).

The ability to use this technology to produce a “look back window” onoverall medication adherence (chronic adherence), without the need touse the system on a daily basis, and still have an accurate picture ofadherence behavior over a defined preceding period of time (hours toweeks) is clinically important and inventive. It reduces subject burdenby eliminating the requirement to use the adherence system on a pill bypill basis as in acute medication adherence monitoring (AMAM). On theother hand, to carry out ideal pharmacometric modeling, most PK expertsfind dose by dose documentation of adherence (yes/no determinations) andthe timing between successive doses (interdose intervals) mostdesirable. An AEM that shows significant absorption in the stomach thatgenerates an EDIM with a longer half life in breath can be easily usedin AMAM, IMAM and/or CMAM modes (see Example 5).

The accumulation factor (Katzung B G, Basic and Clinical Pharmacology,page 39, 6^(th) Edition, 1995, Appleton & Lange, Norwalk, Conn.)predicts the ratio of the steady state concentration of the EDIM inbreath to that following the first dose of AEM.

$\begin{matrix}{{AF} = \frac{1}{F_{Lost}}} & {{Equation}\mspace{14mu} 5}\end{matrix}$

where:

-   AF=accumulation factor; and-   F_(Lost)=fraction lost in one dosage interval (T), prior to the next    dose.

The fraction of the EDIM lost in one dosage interval (T) just before theadministration of the next dose of AEM can be determined fromre-arranging Equations 1 and 3 into the following equation:

$\begin{matrix}{F_{Lost} = {1 - ^{{- 0.693}{(\frac{T}{t\; {1/2}e})}}}} & {{Equation}\mspace{14mu} 6}\end{matrix}$

Thus, the only PK parameter that determines F_(Lost) is the ratio of thedosage interval (T) to the elimination half life (t_(1/2a)). The peak(C_(MAX,SS)) and trough (C_(Trough,SS)) EDIM concentrations at steadystate (4 EDIM half lives to achieve 94% of steady state EDIMconcentration) is equal to peak and trough levels obtained after the1^(st) AEM dose multiplied by the AF. This is shown below:

C _(MAX,SS) =AF*C _(MAX,1st Dose)  Equation 7

C _(Trough,SS) =AF*C _(Trough,1st Dose)  Equation 8

The fraction remaining (F_(Remaining)) simply equal to 1 minus F_(Lost).As illustrated in the below Table A, there is an inverse relationshipbetween the accumulation factor (AF) and the T/t_(e) ratio:

TABLE A Effect of AEM Dosage Interval (T) and EDIM half life (t_(1/2))in Breath on the Accumulation Factor (AF). T (Dosage AccumulationInterval) (hrs) t_(1/2e) (hrs) T/t_(1/2e) F_(Remaining) F_(Lost) Factor(AF) 0.24 24 0.01 0.993 0.007 144.801 1.2 24 0.05 0.966 0.034 29.363 2.424 0.1 0.933 0.067 14.936 4.8 24 0.2 0.871 0.129 7.727 7.2 24 0.3 0.8120.188 5.327 9.6 24 0.4 0.758 0.242 4.131 12 24 0.5 0.707 0.293 3.41514.4 24 0.6 0.660 0.340 2.940 16.8 24 0.7 0.616 0.384 2.602 19.2 24 0.80.574 0.426 2.350 21.6 24 0.9 0.536 0.464 2.155 24 24 1 0.500 0.5002.000 48 24 2 0.250 0.750 1.333 72 24 3 0.125 0.875 1.143 96 24 4 0.0630.937 1.067 120 24 5 0.031 0.969 1.032 144 24 6 0.016 0.984 1.016 168 247 0.008 0.992 1.008 192 24 8 0.004 0.996 1.004 216 24 9 0.002 0.9981.002 240 24 10 0.001 0.999 1.001

In other words, the oral administration of AEMs at frequent intervalsthat generate long half life EDIMs is associated with the greatestdegree of accumulation. For example, if an AEM is orally administeredthat generates an EDIM with an elimination half life of 24 hours(t_(1/2a)=24 hours) on a BID (Q 12 hour basis: T=12 hours), theT/t_(1/2e) ratio is 0.5, and the accumulation factor is 3.415. Thus, thesteady state values of trough (C_(Trough)) and maximum (C_(MAX)) EDIMconcentrations are 3.415× greater than those following the 1^(st) oraldose for an EDIM with a value of T/t equal to 0.5. The time that mustelapse in order for the EDIM to decrease from its steady state values ofC_(Trough) and C_(MAX) to a lower level is readily calculated usingEquation 3, and is used to determine for how long a subject has nottaken their medication.

After the oral administration of the AEM 2-butanol (40 and 60 mg) tohumans, the half life of the EDIM 2-butanone was found to be 11-22 min(Morey T E, et al AIDS Behav, 17(1), 298-306, 2013; Example 4a). Asdemonstrated in this patent disclosure, isopropyl alcohol (IPA) has beenfound to be a promising AEM. The above analysis can be applied to IPA asthe AEM, which rapidly generates the EDIM, acetone, with a longer halflife in breath. For example, Jones-J et al (Anal Toxicol 2000,24(1):8-10) found the mean elimination half life of acetone in humansranged from 17 to 27 hours with an average of 22 hours. We have found,however, that the elimination half life of acetone (d6-acetone) in humanbreath following ingestion of deuterated isopropyl alcohol (d8-IPA) wasbetween 6.4 hours (FIG. 74) and 8.5 hrs (Example 5). Using these 5 halflives of acetone (6.4, 8.5, 17, 22, and 27 hrs) after the ingestion ofIPA at various dosage intervals (T), the following table (Table B)illustrates the following accumulation factors (AF) for acetone inbreath. Similar to what was described above for Table A, theaccumulation factors shown in Table B can be used to determine thelength of time a subject was not adherent to a medication containingIPA, which equals the length of time that elapsed from the expectedoriginal EDIM concentration in breath (e.g., assume C_(Trough,SS) arebeing made) to the concentration measured at a randomly called troughtime, as determined by the EDIM t_(e) Equation 3 by using the EDIM t_(e)and solving for the time (t) to decay from the higher to the lower EDIMbreath concentration:

TABLE B Accumulation Factor (AF) of acetone as a function of variouselimination half lives (t_(1/2e)) and AEM (i.e., isopropyl alcohol, IPA)dosing intervals (T). More frequent dosing with IPA and a longert_(1/2e) lead to greater accumulation. Accumu- AEM T (Dosage lationDosing Interval) t_(1/2e) T/ Factor Interval (hrs) (hrs) t_(1/2e)F_(Remaining) F_(Lost) (AF) QD 24 6.4 3.750 0.074 0.926 1.080 QD 24 8.52.824 0.141 0.859 1.165 QD 24 17 1.412 0.376 0.624 1.602 QD 24 22 1.0910.470 0.530 1.885 QD 24 27 0.889 0.540 0.460 2.174 BID 12 6.4 1.8750.273 0.727 1.375 BID 12 8.5 1.412 0.376 0.624 1.602 BID 12 17 0.7060.613 0.387 2.585 BID 12 22 0.545 0.685 0.315 3.177 BID 12 27 0.4440.735 0.265 3.772 TID 8 6.4 1.250 0.421 0.579 1.726 TID 8 8.5 0.9410.521 0.479 2.087 TID 8 17 0.471 0.722 0.278 3.594 TID 8 22 0.364 0.7770.223 4.489 TID 8 27 0.296 0.814 0.186 5.387 Note: QD (Q 24 hrs), BID (Q12 hrs), and TID (Q 8 hrs) indicates once, twice, and three times perday oral dosing.

Factor 2: Concentration of EDIM in Breath

The concentration of EDIM in breath generated from the administration ofa dose of AEM is an important factor in the overall function of theSMART® Adherence System. The dose of the AEM, because it is quicklyconverted to the EDIM (e.g., 2-butanol and isopropyl alcohol conversionto 2-butanone and acetone, respectively) in the blood plays a pivotalrole in establishing the EDIM concentration in human breath. Theultimate concentration of EDIM in breath will depend on its volume ofdistribution (V_(d)) and the quantity of EDIM liberated from the orallyadministered dose of AEM. Since the V_(d) is fixed in a given person fora given molecular entity, only the AEM dose can be readily altered toincrease or decrease the initial EDIM concentrations attained in breath.

Using the OrbiTrap LC/MS/MS system (FIGS. 60, 61, 72) to measure acetonelevels in real time following ingestion of IPA, the relationship betweenIPA (AEM) dose and the yield of acetone (EDIM) in the breath can bereadily ascertained. For example, as illustrated in FIG. 60, a single100 mg dose of d8-IPA caused an OrbiTrap response that was approximately5.14× greater (peak height: 360,000 versus 70,000) than that caused bythe constant level of background endogenous breath acetone over thecourse of the study. Furthermore, if one assumes that this healthysubject had a typical normal breath acetone concentration of 582 ppb(Diskin A M et al: Physiol Meas 24:107-119, 2003), this would translateto a maximum d6-acetone concentration (C_(MAX)) of 2,993 ppb (=582ppb×5.14) in breath at 3 hours (T_(MAX)) caused by ingesting 100 mg ofd8-IPA. Thus, the background acetone served as an “internal control” forunderstanding the concentration-time relationship of d6-acetone.Assuming dose proportionality (linear PK), if we dosed with the FDAestablished permissible daily exposure (PDE) of IPA (138 mg orally perday), this would have caused an acetone C_(MAX) of 4131 ppb. Note:because the peak response (sensitivity) of the OrbiTrap is the same foracetone and d6-acetone, this approach is technically viable. Also note,as expected, there is an absence of any significant d6-acetone in humanbreath prior to ingestion of the d8-IPA. This is a major advantage ofusing non-ordinary cold isotopologues in this invention. Specifically,it provides the foundation for a SMART® Adherence System with nointerferents, the longest adherence “look back” time window in terms ofassessing chronic adherence (CMAM), and the use of IR sensors withtremendous sensitivity (parts per trillion). Consistent with the yieldof d6-acetone in breath following ingestion of d8-IPA using the OrbiTrapsystem, when a Type 1 (mGC) SMART® device was employed (FIG. 77),similar high concentrations of acetone (2700 to 3000 ppb) in breath werefound after ingesting 100 mg IPA for 5 successive days (FIG. 77).

The maximum safe doses of AEM are well defined by US regulatoryauthorities. For example, the AEMs 2-butanol and IPA, appear to be safeas noted by a number of regulatory agency listings. They are included asdirect food additives in the FDA EAFUS (Everything Added to Food in theUnited States) listing. Likewise, according to the FDA's Q3C Guidancefor Industry, the permissible daily exposure (PDE) for 2-butanol and IPAis 300 and 138 mg/day, respectively. The PDE defines the dose ofcompound that a human can chronically ingest for the rest of their liveswith no regulatory concern. Therefore, from a toxicological perspective,these types of AEMs (e.g., 2-butanol and IPA) are excellent candidatesfor use to document adherence. It should be noted that other classes ofcompounds, including but not limited to sulfur containing molecules,(e.g. allicin (garlic) and dimethyl sulfoxide (DMSO)) are listed in theFDA food database and generate short and long acting metabolites inbreath, which would be suitable for the SMART Adherence System. Forexample, the FDA in its Q3C guidance lists the PDE for DMSO as 50 mg perday. DMSO has an elimination half life of 12-15 hours in humans andgives rise to highly volatile markers such as dimethyl sulfide (DMS).Because these molecules are present in various foods and specificpathological conditions, the use of cold non-ordinary isotopes on thehydrogen, carbon, sulfur, and/or oxygen atoms of these types of AEM isvery promising and would easily distinguish these EDIMs from backgroundinterferents, particularly when a Type 2 (IR) device can measure theseat very low concentration (low ppt), which would not be associated withmalodorous smells.

Factor 3: Limit of Detection (LoD) of the SMART® Device (Type 1 vs 2) tothe EDIM

Another key factor in determining how long the EDIM can be accuratelymeasured in the breath of humans is the limit of detection (LoD) of theSMART® device. For example, in the current configuration, a Type 1SMART® device has a minimum LoD in the low part per billion (1-5 ppb),whereas a Type 2 SMART® device using infrared (IR), including near ormid IR type systems (e.g., cavity ring detection by Picarro, Sunnyvale,Calif.; or tunable lasers by Daylight Solutions, San Diego, Calif.)measurements of isotopologues of volatile compounds in the gas phase(e.g., deuterated water such as DHO) are detectable down to a LoD in theparts per trillion (1-1000 ppt). Naturally, it is not required for thesystem to operate at the LoD, and workable results are achievable in thetens of parts be billion range or higher.

Factor 4: Level of Background Interference to Measurement of the EDIM

The level of any background EDIM in breath or interferents that canmimic the EDIM in breath on the sensor can significantly reduce thelength of the adherence “look back” window period, even if the sensorhas a very low LoD to the EDIM. For example, humans naturally have meanendogenous levels of acetone ranging from 293 to 870 (average of themeans=582) ppb (Diskin A M et al: Physiol Meas 24:107-119, 2003), butcan undergo significant variation between humans and even with anindividual during the day. Because endogenous acetone levels reflect acomplex array of many physiological processes (e.g., lipolysis,circadian rhythms, etc.), the content of this ketone in blood and hencebreath can vary significantly over time within an individual and betweenindividuals.

This finding has two consequences with regard to using IPA as an AEM inthe SMART® Adherence System using a Type 1 SMART® device. First, asignificant fraction of orally ingested IPA (deuterated ornon-deuterated) can be absorbed through the stomach and cause a rapidrise above baseline levels in acetone (see FIGS. 53, 54, 60, 61, 62, 63,69, 71, 76, 77), the levels of which are several multiples of backgroundendogenous acetone when IPA is ingested at doses that are deemed safe.If a baseline acetone level is obtained, IPA can be used for effectiveAMAM. In the time period to obtain the 2 breaths (e.g., or 30 min), noordinary physiological process can increase acetone to those levels.This point is not relevant for d6-IPA because there is no backgroundd6-acetone to contend with in the SMART® Adherence System. Second, incontrast, with CMAM, unless the amount of IPA (not non-ordinaryisotopically labeled IPA) that is given orally produces such a highamount of acetone in the breath, which can be clearly distinguished fromnormal variations in endogenous breath acetone concentrations duringactivities of daily living (ADL), it is quite susceptible to falsepositive and false negatives that limits its utility when used for IMAM(adherence window up to 1 day), because in this case the limiting factorfor the adherence window (C_(EDIM,Limit)) is not sensor sensitivity(Type 1 mGC device has an LoD of 1-5 ppb) but rather the level ofbackground endogenous acetone. This high level of endogenous acetone andthe variability of these levels within and between individuals oversustained periods of time during ADL limits the adherence window “lookback” period to AMAM and/or IMAM; it is not suitable for CMAM. Incontrast, the use of deuterated isopropyl alcohol (d8-IPA) as the AEMproduces deuterated acetone (d6-acetone) that does not suffer from thislimitation (no background levels present), provides a long adherence“look back” window (see below, T_(AdhWindow) and values shown in TableD), and is highly suitable for CMAM, even for longer periods of theadherence window look back time (see Equation 9 below: T_(AdhWindow) andTable D).

With regard to background interference affecting another AEM, 2-butanol(ordinary isotopic form=no cold isotopes used), the concentration of2-butanone in breath is typically very low but occasionally subjectswill have higher levels. In order to maximize the sensitivity,specificity, and accuracy of a 2-butanone-based AMAM SMART® system, itis necessary to include a baseline breath sample to mitigate its effect.A rise in 2-butanone levels, typically 5 ppb or greater in breath, arehighly indicative of adherence (see Example 3, Clinical Studies fordetails). Recall that 2-butanol is suitable for AMAM because 2-butanonehas an elimination half life in breath of 11-22 min. Similar tonon-ordinary cold isotopologues of acetone that are generated whend8-IPA is used as the AEM, the use of d10-2-butanol that generated8-2-butanone eliminates any potential for background interference to2-butanone already present in breath at the start of the adherenceassessment. However, it still does not make it suitable for IMAM or CMAMbecause the elimination half life of 2-butanone is the same for bothcold non-ordinary (e.g., deuterated) or ordinary 2-butanone in humanbreath. For simple molecules like 2-butanol, 2-butanone, IPA, andacetone, isotopic substitutions on their structures with coldnon-ordinary isotopes does not cause significant changes in their PKproperties. Consistent with this statement, FIG. 62 shows the parallelrise (concentration-time relations) of deuterated acetone and ordinaryacetone following the ingestion of a capsule containing both d8-IPA andordinary IPA. In contrast, substitutions on much more complex molecularentities such as the deuterated form of the anti-depressant, paroxetine,can cause changes, albeit somewhat subtle, in its PK properties,including but not limited to its susceptibility to CYP-450 metabolism(Concert Pharmaceuticals, Lexington, Mass.; website:http://www.concertpharma.com/index.html).

Given the four factors just discussed, for a specific EDIM with a givenhalf life in breath, how would one establish the adherence window(T_(AdhWindow) Equation 9), where adherence can be assessed? The timerequired for the EDIM to fall from a specific concentration, termedC_(EDIM,o) such as from its trough (C_(EDIM,Trough)) or peak(C_(EDIM,MAX)) levels, to some limiting concentration, termedC_(EDIM,Limit) will define the maximum “look back” detection window. TheC_(EDIM,Limit) is defined by the greater of two factors: 1) SMART®device sensitivity, or 2) EDIM background interference (e.g., variationin the endogenous levels of acetone if the EDIM is IPA generatedacetone). Major advantages of using cold non-ordinary isotopologues asthe AEMs in the SMART® Adherence System include: 1) they generate EDIMs,which have no background interference, and 2) they can be detected withType 2 SMART® Devices that have outstanding LoDs (part per trillionlevel detection levels). Therefore, the adherence “look back” windowwill be greater when cold non-ordinary isotopologues are used as theAEMs.

Equation 9 (from a re-arranged Equation 3) provides the maximum lengthof time (adherence “look back” window), defined as T_(AdhWindow) that itwill take for an EDIM to decay from an initial level, termed C_(EDIM,o)(e.g., EDIM trough [C_(Trough)] or max [C_(MAX)]) to a limiting EDIMconcentration, termed C_(EDIM,Limit), either due to a device LoDlimitation or a background interferent level.

$\begin{matrix}{T_{AdhWindow} = {\frac{t_{{1/2}e}}{0.693}*{\ln \left( \frac{C_{{EDIM},o}}{C_{{EDIM},{Limit}}} \right)}}} & {{Equation}\mspace{14mu} 9}\end{matrix}$

Table C provides various values of T_(AdhWindow) depending upon thet_(1/2e), C_(EDIM,o) and C_(EDIM,Limit) of the EDIM. Table Dspecifically provides this information using the four elimination halflives (6.4, 8.5, 17, 22, and 27 hrs) reported in the text for the EDIM,acetone:

TABLE C Adherence “Look Back” Window Period, termed T_(AdhWindow), asmeasured in hours for an EDIM. Values were calculated from Equation 9.The relationship between the length of time (hours) that will elapse forthe initial EDIM concentration (C_(EDIM, o)) to decay to the EDIM limitconcentration (C_(EDIM, Limit)) (whatever is greater, either the LoD ofthe sensor or background EDIM levels is applicable), given the EDIM halflife in breath (t_(1/2e)). The C_(EDIM, o) could represent variousconcentrations, but preferably trough EDIM (C_(Trough)), maximum EDIMconcentration (C_(MAX)), and/or some EDIM concentration at a fixed timepost dosing (e.g., 20 or 30 min) during a dosing interval. Decay Time(hrs): EDIM Initial Concentration to EDIM Limit Concentration IniialEDIM EDIM Half Life of EDIM in Human Breath (hrs) Breath Conc Limit Conc0.5 0.75 1 2 4 6 8 12 18 24 100 ppb 10 ppt 6.7 10.0 13.3 26.6 53.2 79.8106.4 159.6 239.4 319.2 100 ppt 5.0 7.5 10.0 19.9 39.8 59.8 79.7 119.5179.3 239.0 1 ppb 3.3 5.0 6.6 13.3 26.6 39.8 53.1 79.7 119.5 159.4 5 ppb2.2 3.2 4.3 8.6 17.3 25.9 34.6 51.8 77.8 103.7 250 ppb 10 ppt 7.3 11.014.6 29.2 58.5 87.7 117.0 175.4 263.2 350.9 100 ppt 5.7 8.5 11.3 22.645.2 67.8 90.4 135.6 203.4 271.2 1 ppb 5.0 7.5 10.0 19.9 39.8 59.8 79.7119.5 179.3 239.0 5 ppb 2.8 4.2 5.6 11.3 22.6 33.8 45.1 67.7 101.5 135.4500 ppb 10 ppt 7.8 11.7 15.6 31.2 62.5 93.7 125.0 187.4 281.2 374.9 100ppt 6.2 9.2 12.3 24.6 49.2 73.8 98.4 147.6 221.4 295.2 1 ppb 4.5 6.7 9.017.9 35.8 53.8 71.7 107.5 161.3 215.0 5 ppb 3.3 5.0 6.6 13.3 26.6 39.853.1 79.7 119.5 159.4 750 ppb 10 ppt 8.1 12.2 16.2 32.4 64.8 97.2 129.6194.4 291.6 388.8 100 ppt 6.4 9.7 12.9 25.8 51.5 77.3 103.0 154.6 231.8309.1 1 ppb 4.8 7.2 9.6 19.1 38.2 57.4 76.5 114.7 172.1 229.4 5 ppb 3.65.4 7.2 14.5 29.0 43.4 57.9 86.9 130.3 173.8 1000 ppb 10 ppt 8.3 12.516.6 33.2 66.5 99.7 133.0 199.4 299.2 398.9 100 ppt 6.7 10.0 13.3 26.653.2 79.8 106.4 159.6 239.4 319.2 1 ppb 5.0 7.5 10.0 19.9 39.8 59.8 79.7119.5 179.3 239.0 5 ppb 3.8 5.7 7.6 15.3 30.6 45.8 61.1 91.7 137.5 183.41500 ppb 10 ppt 8.6 12.9 17.2 34.4 68.8 103.2 137.6 206.4 309.6 412.8100 ppt 6.9 10.4 13.9 27.8 55.5 83.3 111.0 166.6 249.8 333.1 1 ppb 5.37.9 10.6 21.1 42.2 63.4 84.5 126.7 190.1 253.4 5 ppb 4.1 6.2 8.2 16.533.0 49.4 65.9 98.9 148.3 197.8 2000 ppb 10 ppt 8.8 13.2 17.6 35.2 13.2105.7 140.9 211.4 317.1 422.8 100 ppt 7.2 10.7 14.3 28.6 10.7 85.8 114.4171.5 257.3 343.0 1 ppb 5.5 8.2 11.0 21.9 8.2 65.8 87.8 131.6 197.5263.3 5 ppb 4.3 6.5 8.6 17.2 6.5 51.9 68.8 103.8 155.7 207.5

TABLE D Adherence “Look Back” Window Period, termed T_(AdhWindow), asmeasured in hours and days using acetone as the EDIM. Values werecalculated from Equation 9. The relationship between the length of time(hours) that will elapse for the initial acetone (EDIM) concentration(C_(EDIM, o)) to decay to the acetone (EDIM) limit concentration(C_(EDIM, Limit)) (whatever is greater, either the LoD of the sensor orbackground acetone levels is applicable), given the acetone half life inbreath (t_(1/2e)). The C_(EDIM, o) could represent variousconcentrations, but preferably trough EDIM (C_(Trough)), maximum EDIMconcentration (C_(MAX)), and/or some acetone concentration at a fixedtime post dosing (e.g., 20 or 30 min) during a dosing interval. DecayTime (hrs): Acetone (EDIM) Initial Decay Time (days): Acetone (EDIM)Initial Concentration to Acetone Limit Level Concentration to AcetoneLimit Level Iniial Acetone Acetone Half Life of Acetone in Human Breath(hrs) Half Life of Acetone in Human Breath (hrs) Breath Conc Limit Conc6.4 8.5 17 22 27 6.4 8.5 17 22 27 100 ppb 10 ppt 85.1 113.1 226.1 292.6359.1 3.5 4.7 9.4 12.2 15.0 100 ppt 63.7 84.7 169.3 219.1 268.9 2.7 3.57.1 9.1 11.2 1 ppb 42.5 56.4 112.9 146.1 179.3 1.8 2.4 4.7 6.1 7.5 5 ppb27.6 36.7 73.4 95.0 116.6 1.2 1.5 3.1 4.0 4.9 250 ppb 10 ppt 93.6 124.3248.5 321.6 394.7 3.9 5.2 10.4 13.4 16.4 100 ppt 72.3 96.1 192.1 248.6305.1 3.0 4.0 8.0 10.4 12.7 1 ppb 63.7 84.7 169.3 219.1 268.9 2.7 3.57.1 9.1 11.2 5 ppb 36.1 47.9 95.9 124.1 152.3 1.5 2.0 4.0 5.2 6.3 500ppb 10 ppt 100.0 132.8 265.5 343.6 421.7 4.2 5.5 11.1 14.3 17.6 100 ppt78.7 104.6 209.1 270.6 332.1 3.3 4.4 8.7 11.3 13.8 1 ppb 57.3 76.2 152.3197.1 241.9 2.4 3.2 6.3 8.2 10.1 5 ppb 42.5 56.4 112.9 146.1 179.3 1.82.4 4.7 6.1 7.5 750 ppb 10 ppt 103.7 137.7 275.4 356.4 437.4 4.3 5.711.5 14.9 18.2 100 ppt 82.4 109.5 219.0 283.4 347.8 3.4 4.6 9.1 11.814.5 1 ppb 61.2 81.3 162.5 210.3 258.1 2.5 3.4 6.8 8.8 10.8 5 ppb 46.361.5 123.1 159.3 195.5 1.9 2.6 5.1 6.6 8.1 1000 ppb 10 ppt 106.4 141.3282.5 365.6 448.7 4.4 5.9 11.8 15.2 18.7 100 ppt 85.1 113.1 226.1 292.6359.1 3.5 4.7 9.4 12.2 15.0 1 ppb 63.7 84.7 169.3 219.1 268.9 2.7 3.57.1 9.1 11.2 5 ppb 48.9 64.9 129.9 168.1 206.3 2.0 2.7 5.4 7.0 8.6 1500ppb 10 ppt 110.1 146.2 292.4 378.4 464.4 4.6 6.1 12.2 15.8 19.4 100 ppt88.8 118.0 236.0 305.4 374.8 3.7 4.9 9.8 12.7 15.6 1 ppb 67.6 89.8 179.5232.3 285.1 2.8 3.7 7.5 9.7 11.9 5 ppb 52.7 70.0 140.1 181.3 222.5 2.22.9 5.8 7.6 9.3 2000 ppb 10 ppt 105.7 140.9 211.4 317.1 422.8 4.4 5.98.8 13.2 17.6 100 ppt 85.8 114.4 171.5 257.3 343.0 3.6 4.8 7.1 10.7 14.31 ppb 65.8 87.8 131.6 197.5 263.3 2.7 3.7 5.5 8.2 11.0 5 ppb 51.9 68.8103.8 155.7 207.5 2.2 2.9 4.3 6.5 8.6 Note: The values of the acetonebreath elimination half lives (6.4, 8.5, 17, 22, and 27 hrs) included inthe analysis were those discussed in Example 5.

Factor 5: Permeability of the AEM for Stomach Absorption

Because the gastric surface is lined by a thick epithelium, mostcompounds do not have significant absorption through the stomach intothe portal vein and hence the liver. Most therapeutic drugs enter theblood (portal vein) via absorption through the small intestine (e.g.,duodenum). However, following oral ingestion, it is well known thatsimple lower molecular weight compounds, including but not limited toalcohols (e.g., ethanol, 2-butanol, IPA) have a significant fraction(e.g., 25-30%) of their systemic absorption into the body from thestomach (Jones A W. Forensic Sci Rev. 2011; 23:91-136). This finding isconsistent with all the data submitted in this patent disclosure, and isa prerequisite to carry out effective AMAM, where the system ismeasuring definitive adherence on a pill by pill (dose by dose) basis.It is a prerequisite for effective AMAM that the AEM be absorbeddirectly through the gastric wall. This is highly desirable, because thebreath marker (EDIM) appearance is not dependent on duodenal absorption,which in turn is highly dependent on the extremely variable process ofgastric emptying. A number of factors can have a major impact on thetime for gastric emptying, including but not limited to food type,stress, and drugs. In contrast, significant gastric absorption is not arequirement for a molecule to serve as an effective AEM in the settingof IMAM or CMAM.

Examples of Different SMART® Adherence System Embodiments

Since most oral drugs being administered in the current health careenvironment are administered once per day (i.e., QD, or every 24 hours),the examples hereafter mentioned will assume that the therapeutic agent,which is linked to the AEM, is given once per day each morning at 8 am.However, the analysis (equations and tables) listed above also readilyenable methods to provide adherence solutions according to alternateregimens, depending upon specific needs and the clinical environment.These factors include: 1) AMAM, IMAM, and CMAM using those drugs thatare also given orally multiple times per day, including but not limitedto twice (BID), three (TID), or four (QID) times per day; 2) multiplevariations on how the SMART® Adherence System can be designed that usea) combinations of ordinary and/or non-ordinary isotopes in AEM(s) thatgenerate EDIMs with different elimination half lives in breath, and b)two (pre-ingestion of medication containing the AEM [baseline breath]and post-ingestion of medication linked to AEM(s)) or one(post-ingestion of medication linked to AEM(s)) breath samples during anadherence assessment; 3) either one or multiple AEMs that generate oneor multiple EDIMs, respectively, 4) different timing of the breathsampling, relative to T_(MAX), during the dosage interval (T) to ensurethe greatest reliability of the adherence assessment (e.g., ensure thatdeceit is not occurring and to minimize or eliminate any potentialinterferents to SMART® Adherence System function).

The illustrative examples provided herein teach how SMART® AdherenceSystems according to this invention can be readily designed andassembled to provide individual AMAM, IMAM, CMAM capabilities along withcombinations of their capabilities (e.g., AMAM plus CMAM capabilities)employing features that provide different levels of certainty that thesystem is providing accurate adherence assessments (safeguards to ensurethat subjects/patients are not deceiving the system). In addition, whenusing the SMART® Adherence System technology, while it will generally bepreferable to configure the system at the level of the individualsubject or patient, as opposed to using global pharmacokinetic (PK)parameters, those skilled in the art will appreciate that the latter isincluded in this invention. The significant variability known to existin PK parameters (e.g., t_(1/2e), C_(Trough), C_(MAX), T_(MAX), etc.)between individuals can be eliminated by determining key PK parametersat the level of the individual subject/patient. In addition, thisapproach also provides a period for the subject/patient to becomeacclimated with the SMART® Adherence System, which may facilitate properuse of the system later in trials or disease management. However,strategies to employ the SMART® Adherence System in the clinical settingbased on population PK is also described. In addition, as a rule ofthumb, it is preferred to use AEMs containing non-ordinary cold isotopesas part of their molecular structure to generate EDIMs labeled with coldnon-ordinary isotopes, which confer two major advantages: 1) nobackground interference, which provides a longer adherence look backwindow and may obviate the need for a 2^(nd) breath when using theSMART® Adherence System, and 2) a Type 2 (IR-based) SMART® device asdisclosed herein may be implemented that has a lower LoD than the Type 1(GC) SMART® device.

Example 28a

In this example, an initial 1^(st) AEM dose pharmacokinetic (PK)analysis over a period of 0 to a maximum of 24 hours is carried out.From the EDIM breath concentration-response data thus generated, theEDIM elimination half life (t_(1/2a)), time to maximum EDIMconcentration (T_(max)), 1^(st) Dose EDIM C_(Trough), 1^(st) dose EDIMC_(MAX), accumulation factor (AF), EDIM concentration at key times suchas 20 and 30 min post ingestion of the AEM contained in the medication(e.g., C_(20min) or C_(30min)), steady state trough EDIM concentration(C_(Trough/SS)), steady state maximum EDIM concentration (C_(MAX,SS)),and the adherence “look back” window time (T_(AdhWindow)) are allgenerated. Note: To ensure an accurate EDIM t_(1/2a) ideally the lasttime point measured in this 1^(st) dose PK analysis includes the EDIMtrough concentration (i.e., the concentration at the time point justprior to the 2^(nd) dose). The AEM could either consist of ordinary ornon-ordinary cold isotopes, which generate ordinary and non-ordinarycold isotopic labeled EDIMs. The EDIM levels in breath are measuredusing the appropriate SMART® device type as disclosed herein.

In this example shown below, the experimental d6-acetone (EDIM)concentration-time data from FIG. 72 following ingestion of the 1^(st)dose of 100 mg d8-IPA is shown in FIG. 79. The black circles indicatethe actual data points obtained in the subject. This data was curve fitto Equation 1, and PK parameters were determined as described inconnection with FIG. 79. FIG. 80 shows the EDIM breath PK afteringestion of 5 sequential doses of 100 mg d8-IPA that attained steadystate levels after 4-5 doses. In the case of using cold non-ordinaryisotope labeled EDIMs (d6-acetone), the T_(AdhWindow) is totallydependent on the EDIM concentration, elimination half life of thed6-acetone, and the LoD of the SMART® Type 1 (IR) device to d6-acetone.

As shown in FIG. 80, the strategy outlined easily provides an adherencewindow of 4-5 days and is highly suitable for CMAM. In addition, it isalso highly suitable for AMAM, because when a two breath script is usedduring an adherence assessment, the rise in d6-acetone over baselinelevels, even with accumulation (see FIG. 79), would be easily detectedif the measurements were made at values less than T_(MAX) (e.g., 20-30min). The latter strategy of asking subjects to provide a baseline(trough) breath sample and one immediately thereafter at a time prior toT_(MAX), such as 20 or 30 min post-ingestion of the medicationcontaining the AEM (determined by 1^(st) single dose PK), not onlyenables AMAM and IMAM in addition to CMAM, but also has other benefits.Specifically, if the system is used in CMAM mode and the subjects arerandomly called to provide a trough breath sample on random days (e.g.,sample provided immediately before their 8 AM dosing), it would berapidly apparent in a subject who was not adherent, because their troughEDIM levels would be markedly lower than steady state trough EDIM levels(degree of non-adherence provided by T_(AdhWindow)) if the subject didnot try to deceive the system by attempting to ingest the medicationprior to providing the trough breath sample. If the subject did try todeceive the system, such behavior would be detected if the measured EDIMtrough concentration or that at 20 or 30 min post ingestion weresignificantly higher than expected. Likewise, other approaches involvingthe random calling of subjects could be used in CMAM to effectivelydetect non-adherent and/or deceitful behavior in a drug being taken at 8am each day by: 1) having a subject provide two breath samples using afixed time interval (30 min) at times before T_(MAX) but after 8 AM(trough time); the EDIM breath concentration with the 2^(nd) breath mustbe rising relative to the concentration with the 1^(st) breath; 2)having the subject provide two breath samples spaced 20 or 30 min apartbeginning at a time after TMAX, say at 8 PM, approximately 12 hoursafter the medicine was taken (or should have been taken) containing theAEM (pill taken at trough, approximately 8 AM). Under thesecircumstances, the 2^(nd) breath sample must have an EDIM concentrationthat is less than (or roughly constant) that observed with the 1^(st)breath concentration; if the 2^(nd) breath EDIM concentration is greaterthan the 1^(st) breath concentration, it would clearly indicate thesubject just ingested the medication to make it appear he/she wasadherent to the drug regimen; and 3) as shown in FIG. 81, a second AEM,(such as 2-butanol), which generates an EDIM, (2-butanone), which has ashort elimination half life of 11-22 min (presence in breath at typicaldoses is 2-3 hours) could be added, which would clearly identifysituations where the subject is trying to abruptly take the medicationwhen randomly called, when they had not been taking it consistently overthe longer term. For example, randomly calling a subject at 8 PM atnight, who was instructed to ingest a medication given daily at 8 AMthat contains the AEMs d8-IPA and d10-2-butanol, if he/she provided asingle breath sample at 8 PM and it contained both d8-2-butanone andd6-acetone, it would immediately indicate deceptive behavior (since the2-butanone would never be detectable in breath hours after the once perday morning dose). The conclusion is drawn that the subject attempted todeceive the system by acutely taking the medication at around 8 PM thatnight, as opposed to 8 AM daily. In this disclosure, we have taught howmore than one AEM can be used to generate multiple EDIMs, which arereadily detected by sensors (e.g., see Example 4a; FIG. 62, FIG. 64;FIG. 78, FIG. 75a ). This exemplary disclosure teaches those skilled inthe art how the SMART® Adherence System can be slightly modified toprovide highly accurate assessments of medication adherence,particularly when these measurements are coupled to a concurrentbiometric measurement (e.g., subject photograph at the time of provisionof a breath sample).

How would the SMART® Adherence System perform if the subject ingestedordinary IPA, as opposed to d8-IPA, at the same doses? In this case, theacetone concentrations generated as the EDIM from IPA would be additiveto the preexisting concentration of endogenous acetone in breath. Here,the limiting EDIM breath concentration would not be dependent on the LoDof the Type 1 Sensor (GC-based with an LoD=5 ppb) but rather the highand variable concentration of endogenous acetone. For illustrationpurposes, it is assumed that the endogenous concentration of acetone isat the mean range of acetone breath levels (i.e., 582 ppb) found inhumans over a 30 day period (Diskin A M et al, Physiol Meas 24:107-119,2003). Thus, after ingestion of IPA, the acetone levels areapproximately additive (endogenous+IPA-derived) and the acetone C_(MAX)and C_(Trough) levels are 2401 ppb (=582+1819 ppb) and 849 ppb (=582+267ppb), respectively. Thus, using a limiting acetone (EDIM) concentrationof 582 ppt, the T_(AdhWindow) for C_(Trough) and C_(MAX) levels ofacetone according to Equation 9 is 17.4 hrs and 4.6 hrs, respectively.It is immediately apparent that the significant background endogenousacetone levels markedly reduces the effective T_(AdhWindow) usingordinary IPA. With ordinary IPA using C_(MAX), the system could be usedin IMAM but not CMAM mode. Furthermore, because IMAM relies onmeasurements up to a day after ingestion of the medication containingthe AEM, all the factors that can cause acetone breath levels to varyover that 1 day period can negatively impact SMART®. With regard toSMART® system performance, the advantages of using d8-IPA are apparentbased on this disclosure. Accordingly, if a Type 1 (GC-based) SMARTdevice is used for IMAM, it would be ideal to use an ordinaryisotope-based AEM that generates a distinctive EDIM (minimal to nobackground interference; no endogenous levels) that is sensitivelydetected with the sensor.

With CMAM, if a subject is regularly or randomly asked to provide abreath sample to the SMART® device at a particular time in a day (e.g.,time at trough, peak concentration, or 20 to 30 min post ingestion ofthe medication containing the AEM), if his/her EDIM concentration fallswithin their personal EDIM concentration band (preferential approach),they will have been adherent over a period of time determined by PK. Ifthe opposite is true, they are deemed non-adherent, and the length ofnon-adherence is determined by the equations/tables provided herein(based on PK principles). The ingestion of IPA as the AEM, eitherordinary and/or non-ordinary isotopic labeled IPA, can be used for AMAM,IMAM, and/or CMAM.

The discussion above centers around the use of single AEM dose PK,tailored at the level of the individual subject, as being the idealapproach to determine the key PK parameters that enable the use of theSMART® Adherence System. As mentioned previously, this is the preferredapproach. The single dose PK strategy in a given individual allows PKparameters along with their standard errors (and confidence intervals)to be extended to determinations carried out over a number of days in agiven individual to provide even better PK parameters in a particularindividual, which could take into account day to day variability such asfood, etc. However, the premonitory “lead in” period can be abolished ifthe same PK parameters are derived from the 1^(st) dose AEM technique,but it is carried out in a large number of subjects to determine globalpopulation-based PK estimates (see Example 3 for different examples ofthis analysis). These same parameters, as described above, aredetermined using standard statistical approaches and the 90%, 95%, and99% confidence intervals are determined. Depending upon the clinicalcircumstances, the 90%, 95%, or 99% confidence interval global PKparameter ranges are employed to guide use and interpretation of theSMART® Adherence System data generated for individual subjects.

Example 28b

In this example, the trough concentration of EDIM (C_(Trough)) and theconcentration of EDIM at a time post ingestion (e.g., 30 min), termedC_(30min), is determined at each AEM dose over a period of 4-7 days.Using experimentally derived PK parameters and applying them to Equation1, allows the d6-acetone (EDIM) concentration-time relationship to becreated (FIG. 82, top panel). Specifically, FIG. 82 illustrates the EDIM(d6-acetone) concentration-time relationships for the 1^(st) seven dosesof medication containing the AEM (100 mg d8-IPA). The bottom panel ofFIG. 82 illustrates the relationship between d6-acetone C_(Trough)levels against time (AEM doses 1 through 7). This experimental data(bottom panel) was curve fit to the equation shown in the bottom panel,providing an estimate along with their standard errors of the fit of the1^(st) order elimination rate constant (k_(e)) (and hence the EDIMelimination half life (t_(1/2e))) and the steady state EDIM troughconcentration (C_(Trough,SS))—Using Equation 9, as described in thelegend of FIG. 82, by substituting the d6-acetone C_(Trough,SS) value asC_(EDIMo), the d6-acetone elimination half life as the EDIM eliminationhalf life (t_(e)), and the Type 2 sensor cutoff concentration (10 or 100ppt) as the C_(EDIMo,Limit) value, the adherence “look back” window time(T_(Adhwindow)) is readily calculated, which in this case is the time itwould take for the d6-acetone concentration to decay from C_(Trough,SS)to the Type 2 sensor LoD level for d6-acetone. Note: C_(30min) can alsobe measured and used as C_(EDIMo) value, because of the numerousbenefits it can promulgate in terms of system accuracy and preventingsubjects from successfully deceiving the system (see discussion inExample 28a above).

The discussion in Example 28b above centers around the use of serialEDIM C_(Trough) (with or without C_(30min)) measurements to estimate theEDIM elimination half life (t_(1/2e)) at the level of the individualsubject, as being the ideal approach to determine the key PK parametersthat enable the use of the SMART® Adherence System. As mentionedpreviously, this is the preferred approach. The serial EDIM C_(Trough)PK strategy in a given individual allows determination of PK parameters,including EDIM t_(1/2e) and steady state levels of trough EDIMconcentration C_(Trough,SS)). This in turn allows the T_(AdhWindow) tobe determined as described previously, along with their standard errors(and confidence intervals). However, the premonitory “lead in” periodcan be abolished if the same PK parameters derived from the EDIM CTroughstudies at the individual level are now carried out in a large number ofsubjects to determine global population-based PK estimates. These sameparameters, as described above, are determined using standardstatistical approaches to derive the 90%, 95%, and 99% confidenceintervals. Depending upon the clinical circumstances, the 90%, 95%, or99% confidence interval global PK parameter ranges are used to guide useand interpretation of the SMART® Adherence System data generated forindividual subjects.

Example 29 Breath-Based Naltrexone Adherence Tool to ManageNarcotic-Addicted HIV Patients and SMART® Naltrexone Formulation

We found that 1) naltrexone can be formulated with acceptable stabilityin a hard gel capsule in an isotropic solution consisting of 2-butanoland oleic acid, and 2) after oral ingestion in humans, this type ofnaltrexone formulation rapidly and reliably causes a robust increase inthe concentration of breath markers (e.g., 2-butanone) that can beeffectively used to definitively document ingestion of the naltrexonedose form using the SMART Adherence System. Based on these results, weconclude that the pharmaceutical development of a viablethermodynamically stable SMART formulation of naltrexone is highlyfeasible, and the SMART Adherence System can be effectively employed todefinitively document ingestion of the SMART naltrexone formulations.The use of SMART naltrexone formulations in the SMART Adherence Systemholds significant promise to ensure that high risk subjects such asopioid-addicted HIV patients ingest this narcotic receptor antagonist asdirected by their health care provider. This work further confirms theutility of this invention for making a wide variety of SMART medicationformulations for which adherence monitoring is enabled by utilizing theSMART device disclosed herein.

In this study, eight subjects were given four different formulations ina double blind, randomized, crossover manner. These formulations were:

Naltrexone Formulation Base (powder) 2-Butanol Isopropanol Oleic AcidL-Carvone 1 45.2 mg 80 mg (98 uL) — 160 mg (179 uL) — Formulation InsideWhite Size 0 LiCaps Capsule 2 45.2 mg 40 mg (49 uL) 30 mg (38 uL) 160 mg(179 uL) — Formulation Inside White Size 0 LiCaps Capsule 3 45.2 mg 40mg (49 uL) — 160 mg (179 uL) 30 mg (32 uL) Formulation Inside White Size0 LiCaps Capsule 4 45.2 mg 40 mg (49 uL) — — — Naltrexone base powderplaced inside Size 0 LiCap Capsule that also contains a Size 2 LiCapCapsule containing 2-buitanol

All of these formulations contained 45.2 mg of naltrexone base powderand either 40 mg or 80 mg of 2-butanol. Three of these formulations (F1,F2, F3) had the naltrexone and 2-butanol mixed with the excipient (160mg of oleic acid) with either 30 mg of isopropanol or L-carvone, thenplaced inside of a white size 0 LiCaps capsule. One of the formulations(F4) served as a check on the effect that the excipient had on thenaltrexone and 2-butanol by having no excipient present but ratherhaving 40 mg of 2-butanol placed inside of a size 2 LiCap capsule, thenhaving this capsule placed inside of a while size 0 LiCap capsule thatcontained the naltrexone base powder. All formulations were prepared bya certified compounding pharmacy (Westlab Pharmacy, Gainesville, Fla.)on the same day that they were used in the study.

Breath samples taken at time points of ˜5 minutes (used as a blankbreath sample taken prior to pill ingestion), 0 minutes (takenimmediately after pill ingestion), 10, 20, 40, 60, and 90 minutes postpill ingestion were collected by having the subject breath directly intoa mouthpiece attached to a Xhale SMART mGC. Each subject used one ofeight mGCs for the length of the study (the same mGC for each of thefour visit dates), with the mGC initially calibrated for 2-butanone oneday before the study started and again after the last subject wasfinished with the study. The mGC initial and final 2-butanonecalibration results were collected. Calibration standards for acetonewere analyzed after the final calibration standards for 2-butanone wereanalyzed. Breath samples (100 cc) were collected at the 30 minute timepoint post-ingestion onto Markes 3½′×¼″ stainless steel thermaldesorption tube packed with Tenax TA for GC/MS analysis. The GC/MS wascalibrated using the same standards used for the mGC initialcalibrations by collecting 100 cc from the gas standards in the Tedlarbags onto the Markes thermal desorption tubes. All of the Markes TenaxTA tubes for both the standards and samples were stored in arefrigerator located in the laboratory at Xhale Inc. until theiranalysis to provide 2-butanone breath concentration data resulting fromingestion of the four formulations for each subject at each time point.This data shows the average of the results for each formulation, andshows the dose-dependence relationship between the amount of 2-butanolingested and the corresponding concentration of 2-butanone in the breathsamples in this study. There is no statistically significant differencein using an excipient versus having the taggant separated from thenaltrexone base (by using a pill-in-pill design). One of theformulations (F2) used in this study contained 30 mg of isopropylalcohol in addition to the naltrexone base and 2-butanol. Isopropylalcohol is metabolized to acetone in the same manner that 2-butanol ismetabolized to 2-butanone. Since only this formulation containedisopropyl alcohol, breath samples in the subjects who ingested thisformulation should show a statistical increase in breath acetoneconcentrations over baseline levels. Since the acetone standards for thecalibration curve were created in blank breath, which contains asignificant amount of acetone, acetone concentrations were calculatedusing the calibration curve and then normalized to the t=0 time point.The average of the results for each formulation unambiguously shows theincrease in acetone breath concentrations caused by the ingestion of 30mg of isopropyl alcohol. The breath 2-butanone concentrations determinedby collecting a breath sample at the 30 minute time point followed byGC/MS analysis was compared to an average of the 2-butanone breathconcentrations using the Xhale SMART mGCs between the 20 minute and 40minute time points. The 2-butanone breath concentrations obtained usingboth techniques. While the GC/MS retains linearity past 1000 ppb of2-butanone, the mGC has a much smaller linearity range (up to 50 ppb of2-butanone). The higher the breath concentration of 2-butanone, thelower the sensitivity (defined as the slope of the calibration curve)the mGC has for this compound. This loss in sensitivity results in amuch greater precision at high concentrations, which can result in adeviation between values obtained using this instrument versus aresearch-grade GC/MS instrument. The technique used to collect breathsamples analyzed by both instruments (side-stream collection onto a trapcontaining a very small amount of adsorbent for the mGC versus in-linecollection onto a trap containing 300 mg of adsorbent for the GC/MS) canalso lead to a poor correlation between these two analytical techniques,particularly at high concentrations.

The results obtained indicate that the ingestion of isotropicthermodynamically stable formulations of naltrexone containing alcoholssuch as 2-butanol and isopropanol (IPA) reliability and rapidly generatesignificant levels of 2-butanone and acetone above baseline levels,respectively. The formulations contained the appropriate amount of2-butanol and naltrexone, were stable, and showed no evidence ofnaltrexone degradation during storage. Finally, the results indicatethat the SMART system can be effectively employed to definitively detectingestion of the SMART naltrexone formulations, using a pre-established2-butanone cutoff concentration of 5 ppb or greater at early breathsampling times post ingestion. Specifically, every subject, who ingestedF1, F2, F3, and F4 would be have been detected by the SMART system (100%sensitivity).

Example 30 Production and Use of Carbonates for Use as Surface Coatingsor Markings for API Adherence Monitoring

Those skilled in the art will appreciate, based on the followingspecifics, that a wide range of solid forms of the markers (primary orsecondary alcohols or other markers) disclosed herein may bemanufactured for surface coating.

Two different carbonates were synthesized for this work according to thefollowing schemes:

Preparation of Sodium Isopropyl Carbonate:

Isopropanol (1000 mL) was taken lab reactor and added Na metal (6 g).The reaction mixture was heated to 80° C. for 3 h (sodium metaldissolved, clear solution was observed). The reaction mixture was cooledto 0 to 5° C. and purged CO2 (thick suspension was observed after 30min). Aliquot were drawn after 30 min, concentrated and the solidobtained was analyzed by 1H NMR. The reaction mixture was allowed tostir for additional 30 min. The complete reaction mixture was rinsedwith isopropanol (100 mL) and transferred to an R.B flask, concentratedunder vacuum to get white solid. The solid obtained after concentrationwas dried under vacuum at 40° C. at 1 h to get 26 g of white solid.

Sodium isopropoxy carbonate is formed according to this method. The MACfrom isopropanol seems to decompose at 60° C. MAC on treatment withwater decomposes and white solid was isolated. The 13C NMR shows onlyone peak at 161.9 ppm.

Synthesis of Sodium butan-2-yl Carbonate:

Synthesis 1: Three grams of Na metal was dissolved in 1000 mL ofanhydrous 2-butanol at 90° C. The solution was cooled to between 0 and5° C. and purged with CO₂ for 45 minutes. The resulting suspension wasconcentrated under high vacuum at 40-50° C. to obtain 15 g of whitesolid. ¹H-NMR: δ ppm (in D2O)=0.89 (triplet, 3H), 1.17 (doublet, 3H),1.50 (multiplet, 2H), 4.38 (multiplet, 1H) ¹³C-NMR: δ ppm (in D2O)=9.48,19.59, 28.97, 73.96 and 159.61. IR: ν (cm⁻¹)=1651, 1458, 1369 and 1289(indicative of an alkyl carboxylate).

Synthesis 2: Six grams of Na metal was dissolved in 100 mL of anhydrousisopropanol at 80° C. The solution was cooled to between 0 and 5° C. andpurged with CO₂ for 60 minutes. The resulting suspension wasconcentrated under high vacuum at 45° C. to obtain 26 g of white solid.¹H-NMR: δ ppm (in D2O)=1.17 (doublet, 6H), 4.53 (multiplet, 1H)¹³C-NMR:δ ppm (in D2O)=21.89, 69.13 and 159.26. IR: ν (cm⁻¹)=1650, 1458, 1369and 1292 (indicative of an alkyl carboxylate).

The butan-2-yl carbonate and Sodium isopropyl carbonate were eachseparately included in medication capsules, and ingested. The carbonatequickly is converted in vivo into the cognate alcohol and that isquickly metabolized, in the case of the sodium isopropyl carbonate, intoacetone and, in the case of the butan-2-yl carbonate, into the butanone.These ketones were quickly detected in the exhaled breath, as shown inFigures

API Surface Coating and Release of EBM on Ingestion:

Having demonstrated that the carbonate form of an alcohol, which is asolid at room temperature, is quickly and efficiently liberated in theexhaled breath as the desired EBM, those skilled in the art know how toformulate powders for surface coating or marking of API's. Inparticular, about 10 to 100 mg of the carbonate is formulated with adessicant and flow promoter for dispensation onto the surface of adosage form. The surface coating or marking is preferably deposited ontothe surface of the API at a position where a barrier film separates thesurface coating from the API and this surface coating is preferablyover-coated by a barrier to prevent moisture from seeping into thesurface coating, and/or to prevent any loss of the surface coating tothe atmosphere or from being abraded during packaging, shipping orhandling. In a highly preferred embodiment, a user of the surface coatedAPI would have no way of knowing that the surface coating includes amarker which, when the API is ingested, is quickly released and evolvesthe EBM. Where one or more non-ordinary isotope(s) is/are included inthe surface coating, e.g. in the isopropyl or butyl carbonate, evensmaller quantities of the marker may be included—e.g. 0.001 to 50 mg,alternatively 0.01 to 25 mg, alternatively 0.1 to 15 mg, and mostpreferably, anywhere from about 1 to 20 mg is sufficient to produce areadily detectable evolution of non-ordinary isotope containing EBM. Inanother embodiment according to this aspect of the invention, the markerpowder is encapsulated and included in an encapsulated state in a fillformulation in a capsule with an API.

Those skilled in the art, based on these examples, would appreciate thatsimilar syntheses carried out with more complex secondary alcohols orprimary alcohols produce carbonates with varying properties andabilities to act as EDIMs to produce EBMs. Likewise, other metal saltsof these carbonates find utility according to the present disclosure invarious contexts as needed.

What is claimed is: 1.-35. (canceled)
 36. An apparatus for identifyingand/or quantitating volatile compounds in a gas sample comprising: atleast one sensor adapted for identification and/or quantitation of avolatile compound of interest present in said gas sample; and at leastone capture device which releasably captures volatile compounds in saidgas sample; said apparatus further comprising a catalytic incineratorbetween said at least one capture device and said at least one sensorwhich converts said volatile compound of interest to carbon dioxide andwater prior to contact with said at least one sensor.
 37. The apparatusof claim 36, wherein the capture device does not capture moisture,hydrogen, nitrogen, or carbon dioxide, in said gas sample, and whichreleases captured volatile compounds for sensing by said at least onesensor;
 38. The apparatus according to claim 36 adapted for identifyingand/or quantitating volatile compounds wherein said gas sample iscomprised by exhaled breath of a subject, said apparatus furthercomprising at least one or a combination of: a. at least one biometriccapture device for concurrent capture of a biometric specific to saidsubject when said gas sample is provided by said subject to saidapparatus; b. a mouthpiece for delivery of an exhaled breath sample bysaid subject to said apparatus, said mouthpiece being operativelycoupled with an exhaled breath detection sensor; and c. a push button onsaid apparatus that said subject can press to self-report adherence to adose of a medication.
 39. The apparatus according to claim 37 comprisingat least two sensors with differential sensitivity to a volatilecompound of interest in the exhaled breath of a subject.
 40. Theapparatus according to claim 36 comprising said catalytic incineratorwherein said at least one sensor is an infrared sensor adapted to detectwater, carbon dioxide or both water and carbon dioxide containingnon-ordinary but stable isotopes of carbon, oxygen or hydrogen.